Deuterated fluorophores

ABSTRACT

The present invention is generally directed to the synthesis and use of fluorophores. It is more specifically directed to the synthesis and use of deuterated fluorophores. In one case, the present invention provides a compound of the structure shown in FIG. 44.

This application claims the priority benefit of U.S. Provisional PatentApplication No. 62/762,987, filed May 29, 2018. The content of thisapplication is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention is generally directed to the synthesis and use offluorophores. It is more specifically directed to the synthesis and useof deuterated fluorophores.

BACKGROUND OF THE INVENTION

Fluorescent compounds can be used as covalent or noncovalent labels toimpart fluorescence to a sample. A critical characteristic offluorescent labels is the number of photons they emit, which relates totheir brightness and photostability. Improving brightness andphotostability is essential for increasing the sensitivity ofmeasurements involving fluorescence. There have been literature reportsregarding fluorescent compounds, especially those with improvedfluorescence properties.

For instance, U.S. Pat. No. 6,130,101, entitled “Sulfonated xanthenederivatives” is allegedly directed to the following: “The presentinvention describes xanthene dyes, including rhodamines, rhodols andfluoresceins that are substituted one or more times by a sulfonic acidor a salt of a sulfonic acid. The dyes of the invention, includingchemically reactive dyes and dye-conjugates are useful as fluorescentprobes, particularly in biological samples.” Abstract.

U.S. Pat. No. 6,184,379, entitled “Rhodamine derivatives and the usethereof” is allegedly directed to the following: “The invention concernsrhodamine derivatives of the general formulae [shown] in which Ca-Cdeach denote a C atom, and Ca and Cb as well as Cc and Cd are eitherlinked together by a single bond or by a double bond; X1 to X16 denoteindependently of one another halogen, sulfonic acid, hydrogen or analkyl residue with 1-20 C atoms in which the alkyl residue can besubstituted with one or several halogen or sulfonic acid residues; R1and R2 are either identical or different and denote either hydrogen,alkyl with 1-20 C atoms, polyoxyhydrocarbyl units, phenyl or phenylalkylwith 1-3 carbon atoms in the alkyl chain in which the alkyl and/orphenyl residues can be substituted by one or several hydroxy, halogen,sulfonic acid, amino, carboxy or alkoxycarbonyl groups where alkoxy canhave 1-4 carbon atoms, R1 contains at least one activatable group, R2and X4 can be optionally linked together via a bridge composed of 0-2 Catoms. In contrast to the prior art, these compounds are characterizedin that A1, A2 and A3 can independently of one another denote hydrogen,cyano, halogen and sulfonic acid; B1 denotes either halogen, cyano orhydrogen; B2 denotes hydrogen, amide, halogen and an alkyl residue with1-20 C atoms. In addition, the invention concerns activated rhodaminederivatives, correspondingly conjugated biomolecules and their use indiagnostic systems.” Abstract.

U.S. Pat. No. 8,580,579, entitled “Hydrophilic and lipophilic rhodaminesfor labelling and imaging” is allegedly directed to the following: “Theinvention relates to novel and improved photostable rhodamine dyes ofthe general structural formulae I or II and their uses as fluorescentmarkers, e.g. for immunostainings and spectroscopic and microscopicapplications, in particular in conventional and stimulated emissiondepletion (STED) microscopy and fluorescence correlation spectroscopy.The partially deuterated analogues are useful as molecular massdistribution tags in mass spectroscopic applications, wherein R₁=anunsubstituted or substituted alkyl group, including a cycloalkyl group,or heterocycloalkyl group; R₂═H, an unsubstituted or substituted alkylgroup, including a cycloalkyl group, or heterocycloalkyl group, or anunsubstituted or substituted aryl group or heteroaryl group, or anycombination of such groups; X═CH₂, C═O, C═NOR^(a), C═NNR^(a)NR^(b),CH(OR^(a)), O, S, SO, SO₂, or any other derivatives of these groups,with Ra and Rb independently being H or an organic residue, inparticular an unsubstituted or substituted (cyclo)alkyl group orheterocycloalkyl group, an unsubstituted or substituted aryl group orheteroaryl group; Z=a negatively charged group with 1, 2, 3, 4 or 5charges per anion.” Abstract.

Despite these reports, there is still a need in the art for novelfluorophores and their use in various methods.

BRIEF DESCRIPTION OF THE INVENTION

In one case, the present invention provides a compound of the followingstructure:

wherein R₁ is independently selected from halogen, H, D, CN, OH,O(alkyl), O(aryl), SH, S(alkyl), S(aryl), NH₂, N(alkyl), N(aryl), NO₂,CHO, C(O)alkyl, C(O)aryl, COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl),C(O)NH(aryl), PO₃H₂, SO₃H, alkyl, substituted alkyl, aryl, substitutedaryl, alkenyl, substituted alkenyl, or where the R₁ and R₁′substituents, taken together with the carbon atoms to which they arebonded, form a substituted or unsubstituted cycloalkyl or cycloalkenylring containing 3, 4, 5, 6, 7, 8, or 9 carbon atoms; R₁′ isindependently selected from halogen, H, D, CN, OH, O(alkyl), O(aryl),SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃, NH(aryl),NH(aryl)₂, NO₂, CHO, COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl),C(O)NH(aryl), PO₃H₂, SO₃H, alkyl, substituted alkyl, aryl, substitutedaryl, alkenyl, substituted alkenyl, and only one of R₁ and R₁′ can be Dwhen X is CF₃, and only one of R₁ and R₁′ can be CD₃ when Q is O; R₂, R₃and R₄ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃,NH(aryl), NH(aryl)₂, NO₂, CHO, COOH, COO(alkyl), COO(aryl),C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl and substituted alkyl,aryl, substituted aryl, alkenyl, substituted alkenyl; Q is selected fromC(alkyl), C(alkyl)₂, NH, N(alkyl), O, S, Si(alkyl)₂, SO₂, P(O)(alkyl),P(O)(aryl), PO₂H, and Se; W is selected from C and N; X is selected fromH, D, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl,substituted alkenyl, halogen, CN, O, OH, O(alkyl), O(aryl), SH,S(alkyl), S(aryl), NH₂, N(alkyl), N(alkyl)₂, N(aryl), N(aryl)₂, NO₂,CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl), COO(aryl), PO₃H₂ andSO₃H; Y is selected from H, D, C(alkyl), C(aryl), C(alkenyl), C(alkyl)₂,NH₂, NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, O and S; Z is selectedfrom H, D, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl),NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂, CHO,C(O)alkyl, C(O)aryl, COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl),C(O)NH(aryl), PO₃H₂, SO₃H, alkyl, substituted alkyl, aryl, substitutedaryl, alkenyl or substituted alkenyl, or Z and Y, taken together withthe carbon atoms to which they are bonded, form a substituted orunsubstituted cycloalkyl or cycloalkenyl ring containing 4, 5, 6, 7 or 8ring carbon atoms, or Z and Y, taken together with the carbon atoms towhich they are bonded, form a substituted or unsubstituted aryl ring.

In another case, the present invention provides a compound of thefollowing structure:

wherein R₁ is independently selected from halogen, H, D, CN, OH,O(alkyl), O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂,N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂, CHO, COOH, COO(alkyl), COO(aryl),C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl and substituted alkyl orwhere the R₁ and R₁′ substituents, taken together with the carbon atomsto which they are bonded, form a substituted or unsubstituted cycloalkylring containing 3, 4, 5, 6, 7, 8, or 9 carbon atoms;

R₁′ is independently selected from halogen, H, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃,NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH,COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyland substituted alkyl, and only one of R₁ and R₁′ can be D when X isCF₃, and only one of R₁ and R₁′ can be CD₃; R₂, R₃ and R₄ areindependently selected from H, halogen, D, CN, OH, O(alkyl), O(aryl),SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl; Q is selected from C(alkyl)₂, N(alkyl), O, S,Si(alkyl)₂, SO₂, P(O)(alkyl), P(O)(aryl) and Se;

W is selected from C and N; X is selected from H, alkyl, substitutedalkyl, aryl, substituted aryl, halogen, CN, OH, O(alkyl), SH, S(alkyl),S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂,CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl), COO(aryl), PO₃H₂ andSO₃H; Y is selected from H, C(alkyl)₂, N(alkyl), N(alkyl)₂, O and S; Zis selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂,CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl), COO(aryl),C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl, and substituted alkylor Z and Y, taken together with the carbon atoms to which they arebonded, form a substituted or unsubstituted cycloalkyl ring containing4, 5, 6, 7 or 8 ring carbon atoms, or Z and Y, taken together with thecarbon atoms to which they are bonded, form a substituted orunsubstituted aryl ring.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Jablonski diagram for and excited state reactions ofrhodamine dyes.

FIG. 2 shows quantum yield values of polycyclic aromatic compounds 5-7and their deuterated analogs.

FIG. 3 shows the quantum yield value of porphyrin 8 and its deuteratedanalog.

FIG. 4 shows quantum yield values of rhodamine 9 and its deuteratedanalog.

FIG. 5 shows previous examples of fluorophores with deuterated N-alkylgroups 10-12.

FIG. 6 shows synthesis of dyes 1, 14-22 and their deuterated analogsfrom fluorescein ditriflate 13 and their spectral properties.

FIG. 7 shows absorbance (abs) and fluorescence emission (em) spectra of1 and 14 in 10 mM HEPES pH 7.3.

FIG. 8 shows photobleaching of 1 and 14 in 10 mM HEPES pH 7.3.

FIG. 9 shows a comparison of the bleaching time constants (s) ofdifferent rhodamines (1, 15, 17) and their deuterated analogs (14, 16,18).

FIG. 10 shows a comparison of the intrinsic brightness (ε×Φ) ofrhodamine dyes 1, 14-22 in 10 mM HEPES pH 7.3.

FIG. 11 shows the absorbance (abs) and fluorescence emission (em)spectra of 17 and 18 in 10 mM HEPES pH 7.3.

FIG. 12 shows the absorbance (abs) and fluorescence emission (em)spectra of 19 and 20 in 10 mM HEPES pH 7.3.

FIG. 13 shows the absorbance (abs) and fluorescence emission (em)spectra of 21 and 22 in 10 mM HEPES pH 7.3.

FIG. 14 shows the photobleaching of 17 and 18 in 10 mM HEPES pH 7.3.

FIG. 15 shows the photobleaching of 15 and 16 in 10 mM HEPES pH 7.3.

FIG. 16 shows the absorbance (abs) and fluorescence emission (em)spectra of 15 and 16 in 10 mM HEPES pH 7.3.

FIG. 17 shows the relative singlet oxygen quantum yield (¹O₂) of 15 and16.

FIG. 18 shows the normalized fluorescence emission spectra and zoom-inof 15 taken periodically during photobleaching experiments showing 9 nmblue-shift due to dealkylation.

FIG. 19 shows the normalized fluorescence emission spectra and zoom-inof 16 taken periodically during photobleaching experiments showing 4 nmblue-shift due to dealkylation.

FIG. 20 shows the chemical structures of HaloTag ligand 23 anddeuterated analog 24.

FIG. 21 shows the fluorescence quantum yield of HaloTag-bound 23 anddeuterated analog 24.

FIG. 22 shows the fluorescence lifetime of histone H2B-HaloTag-bound 23and deuterated analog 24 in live cells measured using FLIM.

FIG. 23 shows the relative singlet oxygen quantum yield (¹O₂) ofHaloTag-bound 23 and deuterated analog 24.

FIG. 24 shows the image of the nucleus of a mammalian cell expressinghistone H2B-HaloTag fusion and incubated with 23 (10 pM, 30 min), thenwashed for 30 minutes prior to fixation and imaging.

FIG. 25 shows the image of the nucleus of a mammalian cell expressinghistone H2B-HaloTag fusion and incubated with 24 (10 pM, 30 min), thenwashed for 30 minutes prior to fixation and imaging.

FIG. 26 shows the chemical structure of deuterated pyrrolidinyl HaloTagligand 25.

FIG. 27 shows the image of the nucleus of a mammalian cell expressinghistone H2B-HaloTag fusion and incubated with 25 (10 pM, 30 min), thenwashed for 30 minutes prior to fixation and imaging.

FIG. 28 shows the photostability (track lengths, s) of single moleculesof HaloTag ligands 23-25 in live-cell single-particle trackingexperiments.

FIG. 29 shows the brightness (kcps) of single molecules of HaloTagligands 23-25 in live-cell single-particle tracking experiments.

FIG. 30 shows the chemical structures and spectral properties of dyes26-33 in 10 mM HEPES pH 7.3.

FIG. 31 shows the comparison of the intrinsic brightness (ε×Φ) ofrhodamine dyes 26-33 in 10 mM HEPES pH 7.3.

FIG. 32 shows the chemical structures of carborhodamine HaloTag ligands34 and 35.

FIG. 33 shows the chemical structures of Si-rhodamine HaloTag ligand 36and deuterated analog 37.

FIG. 34 shows the fluorescence quantum yield of HaloTag-bound 36 anddeuterated analog 37.

FIG. 35 shows the fluorescence lifetime of histone H2B-HaloTag-bound 36and deuterated analog 37 in live cells measured using FLIM.

FIG. 36 shows the relative singlet oxygen quantum yield (1O2) ofHaloTag-bound 36 and deuterated analog 37.

FIG. 37 shows the image of the nucleus of a mammalian cell expressinghistone H2B-HaloTag fusion and incubated with 36 (10 pM, 30 min), thenwashed for 30 minutes prior to fixation and imaging.

FIG. 38 shows the image of the nucleus of a mammalian cell expressinghistone H2B-HaloTag fusion and incubated with 37 (10 pM, 30 min), thenwashed for 30 minutes prior to fixation and imaging.

FIG. 39 shows the chemical structure of deuterated pyrrolidinyl HaloTagligand 38.

FIG. 40 shows the image of the nucleus of a mammalian cell expressinghistone H2B-HaloTag fusion and incubated with 38 (10 pM, 30 min), thenwashed for 30 minutes prior to fixation and imaging.

FIG. 41 shows the photostability (track lengths, s) of single moleculesof HaloTag ligands 36-38 in live-cell single-particle trackingexperiments.

FIG. 42 shows the brightness (kcps) of single molecules of HaloTagligands 36-38 in live-cell single-particle tracking experiments.

FIG. 43 shows the chemical structures and spectral properties of dyes39-44 in 10 mM HEPES pH 7.3.

FIG. 44 shows a general structure for a compound of the presentinvention.

FIG. 45 shows further structures for a compound of the presentinvention.

FIG. 46 shows further structures for a compound of the presentinvention.

FIG. 47 shows further structures for a compound of the presentinvention.

FIG. 48 shows further structures for a compound of the presentinvention.

FIG. 49 shows further structures for a compound of the presentinvention.

FIG. 50 shows further structures for a compound of the presentinvention.

FIG. 51 shows further structures for a compound of the presentinvention.

FIG. 52 shows further structures for a compound of the presentinvention.

FIG. 53 shows further structures for a compound of the presentinvention.

FIG. 54 shows further structures for a compound of the presentinvention.

FIG. 55 shows further structures for a compound of the presentinvention.

FIG. 56 shows further structures for a compound of the presentinvention.

FIG. 57 shows further structures for a compound of the presentinvention.

FIG. 58 shows further structures for a compound of the presentinvention.

FIG. 59 shows further structures for a compound of the presentinvention.

FIG. 60 shows further structures for a compound of the presentinvention.

FIG. 61 shows further structures for a compound of the presentinvention.

FIG. 62 shows further structures for a compound of the presentinvention.

FIG. 63 shows further structures for a compound of the presentinvention.

FIG. 64 shows further structures for a compound of the presentinvention.

FIG. 65 shows further structures for a compound of the presentinvention.

FIG. 66 shows further structures for a compound of the presentinvention.

FIG. 67 shows further structures for a compound of the presentinvention.

FIG. 68 shows a scheme for synthesizing compounds of the presentinvention.

FIG. 69 shows a further scheme for synthesizing compounds of the presentinvention.

FIG. 70 shows a further scheme for synthesizing compounds of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

Rhodamine dyes such as tetramethylrhodamine (TMR, 1, FIG. 1) remain inwide use due to their excellent brightness, superb photostability, andbroad spectral range.¹⁻³ The photophysics of rhodamines are wellunderstood due to their importance as biological probes and laser dyes.⁴Absorption of a photon excites the TMR molecule from the ground state(1-S₀) ultimately to the first excited state (1-S₁). After excitation,the molecule can relax back to S₀ through a variety of processes.Emission of a photon (fluorescence) competes with nonradiative decaypathways such as twisted internal charge transfer (TICT) where electrontransfer from the aniline nitrogen to the xanthene system gives acharge-separated species (1-TICT) that rapidly decays back to the groundstate (1-S₀) without emitting a photon.⁵ This process competes withfluorescence, thereby decreasing fluorescence quantum yield (Φ).Alternatively, the excited dye can undergo intersystem crossing to thefirst triplet excited state (1-T₁) where it can sensitize singlet oxygen(¹O₂), returning to the ground state (1-S₀). The resulting ¹O₂ can thenreact with the ground state of the dye, oxidizing the aniline nitrogento radical cation (2), which can undergo deprotonation to acarbon-centered radical (3) that ultimately results in dealkylation ofthe dye to form trimethylrhodamine (4; FIG. 1).⁴ This process results ina blue-shift in absorption (λ_(max)) and emission (λ_(cm)) maxima, whichcan complicate multicolor experiments and is a prelude to additionaldealkylation and irreversible photobleaching steps.

Both of these undesirable processes—TICT and dealkylation—can bemitigated through modifications in the chemical structure of the dye.Since both involve oxidation of the aniline nitrogen, methods toincrease the ionization potential of this atom can improve bothbrightness and photostability. It was discovered that replacing theN,N-dimethylamino groups with 4-membered azetidine rings furtherimproved the brightness and photostability of rhodamine and other dyes.This was likely due, in part, to the higher ionization potential ofazetidines, which would make both the TICT and dealkylation pathways(FIG. 1) less favorable.

Another strategy to increase brightness and photostability ofsmall-molecule fluorophores such as 1 was envisioned by replacing thehydrogen (H) atoms on the N-alkyl groups with deuterium (D). Deuteratedalkylamines exhibit higher ionization potentials relative to theirhydrogen-containing analogs,⁶ suggesting that deuteration could decreasethe efficiency of the TICT process and therefore increase quantum yield.This higher ionization potential could also slow the initial electronabstraction step (i.e., 1→42, FIG. 1) and the stronger C-D bond couldslow the rate of deprotonation (i.e., 2→3, FIG. 1), together decreasingthe efficiency of the dealkylation step.

Deuteration has long been proposed as a means to increase Φ⁷ and manyfluorophores show improvements in brightness and photostability indeuterated solvents. Nevertheless, prior examples of deuterated dyes arerare and deuteration typically has a negative or neutral effect on Φ asdemonstrated for simple polycyclic aromatic compounds (5-7; FIG. 2)⁸⁻¹⁰,porphyrins (8; FIG. 3), and rhodamines deuterated at other positions (9;FIG. 4). It was therefore nonobvious that deuteration of the N-alkylgroups would improve the properties of rhodamines. Furthermore, the onlyprevious example of fluorophores with N-alkyl groups deuterated alpha tothe nitrogen are a partially deuterated tetraethylrhodamine-d₁₀ (i.e.,rhodamine B-d₁₀, 10; FIG. 5) and the fully deuteratedtetraethylrhodamine-d₂₀ (i.e., rhodamine B-d₂₀, 11; FIG. 5), which wereprepared to study fragmentation of rhodamines in mass spectrometryexperiments,^(11,12) and the N,N-dimethylaminocoumarin-d₆ compound 12(FIG. 5) which was a control for excited-state proton transferexperiments.¹³ The spectroscopic properties of fluorophores 10-12 werenot reported so the effect of deuterated alkylamino groups onfluorescence was unknown.

The hypothesis that deuteration of N-alkyl groups would improvebrightness and photostability was tested by synthesizing a series ofrhodamine dyes and their deuterated counterparts using a cross-couplingapproach starting from fluorescein ditriflate (13; FIG. 6).¹⁴ We firstcompared TMR (1) and its deuterated analog 14, finding remarkablysimilar absorption maximum (λ_(max)) and fluorescence emission maximum(λ_(cm); FIG. 6) for the two dyes with no change in the shape of theabsorption peak (FIG. 7). Deuteration did affect the brightness andphotostability of the dye, however, with 14 showing a ˜20% increase inboth the extinction coefficient at λ_(max) (ε) and Φ compared to 1 (FIG.6) and slower rate of photobleaching (FIG. 8, FIG. 9). Based on thisresult with TMR (1) other matched pairs of rhodamine dyes with H- orD-containing cyclic N-alkyl groups were tested (15-22, FIG. 6). Like theTMR compounds 1 and 14, increases in ε and Φ for the deuteratedpyrrolidine-, piperidine-, and morpholine-containing rhodamines 18, 20,and 22 were observed compared to the parent compounds 17, 19, and 21(FIG. 6, FIG. 10) with no change in spectral shape (FIG. 11, FIG. 12,FIG. 13); the deuterated pyrrolidine 18 also showed improved in vitrophotostability (FIG. 9, FIG. 14). Interestingly, the deuteratedazetidine-containing rhodamine (16) showed no improvement in ε, Φ, orphotostability over the parent nondeuterated 15 (‘Janelia Fluor’ 549,⁵JF₅₄₉; FIG. 6, FIG. 9, FIG. 10, FIG. 15) although it showed similarspectra (FIG. 16). This result suggests that the azetidine and deuteriumsubstitutions suppress the same nonradiative pathways (e.g., TICT) andare therefore not additive. Interestingly, the deuterium substitutiondid elicit a significantly lower singlet oxygen (¹O₂) quantum yield(FIG. 17), suggesting that deuterium modulates either the intersystemcrossing to T₁ or relaxation from T₁ (FIG. 1). Importantly, both thedeuterated azetidine and deuterated pyrrolidine containing dyes 18 and20 showed higher ‘chromostability’ during bleaching compared tonondeuterated compounds 15 and 17 (FIG. 18, FIG. 19), which indicates ahigher resistance to the undesirable dealkylation pathway (FIG. 1) andconcomitant blue spectral shift.

These dyes were then tested as protein conjugates in vitro and in livingcells. As both azetidine compounds showed high ε and 101, the HaloTag¹⁵ligands of the azetidinyl-rhodamines 15 and 16 (23 and 24; FIG. 20) weresynthesized first. The fluorescence properties of azetidinyl compounds23 and 24 attached to HaloTag protein were compared, finding that thatthe conjugates of the deuterated dye 24 showed higher fluorescencequantum yield in vitro (FIG. 21) and longer fluorescence lifetime incells (τ; FIG. 22) compared to the nondeuterated 23. This resultsuggests that deuteration suppresses a protein-bound-specific mode ofnonradiative decay. Like the free dyes, deuteration also significantlysuppressed ¹O₂ generation (FIG. 23). Both ligands could labelHaloTag-histone H2B fusions in living cells (FIG. 24, FIG. 25).

Based on the high brightness of the deuterated pyrrolidine-containingrhodamine 18 (FIG. 6) the HaloTag ligand of this compound (25; FIG. 26)was also synthesized, which was an excellent live-cell label (FIG. 27).HaloTag ligand 24-26 in live-cell single-particle tracking experimentsusing sparsely expressed Sox2-HaloTag fusions were compared.¹⁶ In linewith the in vitro bleaching experiments on the free dyes (FIG. 9, FIG.14, FIG. 15), deuteration of the azetidine showed no improvement inphotostability in cells (i.e., average track length of individualmolecules; FIG. 28), but the deuterated pyrrolidine rhodamine ligand 25did show significantly longer tracks compared to azetidinyl dyes 23 and24. Deuteration did elicit a higher brightness (i.e., photons/s, FIG.29) with both 24 and 25 conjugates emitting more photons per unit timecompared to conjugates of 23 under equivalent imaging conditions.

This modification was then applied to other rhodamine analogs, focusingon the azetidine and pyrrolidine modifications based on the highbrightness observed for the rhodamines 16 and 18 (FIG. 6). For both theazetidinyl and pyrrolidinyl carborhodamines¹⁷ 26 and 28, substantialincreases in both Φ and ε were observed when the cyclic amines weredeuterated to give 27 and 29 (FIG. 30, FIG. 31). The HaloTag ligands ofcompounds 27 and 29 (34 and 35, FIG. 32) were prepared. TheSi-rhodamines 30 and 32 and their deuterated analogs 31 and 33 (FIG. 30)were then examined. As with the rhodamine series, deuteration of theazetidinyl rhodamine 30 to give 31 did not elicit a large increase influorescence quantum yield (Φ), although the extinction coefficient inwater (ε) was modestly increased, yielding an increase in intrinsicbrightness (FIG. 31). Deuteration of the pyrrolidine-containingrhodamine 32 to give 33 did elicit a substantial increase in both Φ andε, in line with the rhodamine series (FIG. 6).

The HaloTag ligands of the azetidinyl Si-rhodamine compounds (36 and 37,FIG. 33) were then synthesized, and the effect of deuteration in vitroand in living cells was measured. Like the analogous rhodaminecompounds, the deuterated azetidinyl dye 37 showed a substantialincrease in Φ compared to 36 when attached to the HaloTag (FIG. 34);compound 37 also showed increased fluorescence lifetime (τ) as theHaloTag conjugate inside live cells (FIG. 35) and lower ¹ O₂ generation(FIG. 36). Both these dyes were suitable labels for HaloTag expressed incells (FIG. 37, FIG. 38). The deuterated pyrrolidinyl Si-rhodamineHaloTag ligand (38, FIG. 39) was also prepared, which showed improvedcellular labeling (FIG. 40) relative to azetidinyl compounds 36 and 37(cf. FIG. 37 and FIG. 38). In live-cell single-molecule experiments itwas discovered that the HaloTag conjugates of deuterated dyes 37 and 38were both more photostable than the conjugates of nondeuterated 36, withdeuterated pyrrolidine Si-rhodamine 38 showing the longest averagesingle-molecule track length (FIG. 41). Likewise, the conjugates of thedeuterated dyes exhibited higher brightness with 38 showing the highestphotons/s (FIG. 42).

The deuterium substitution was applied to other dyes beyondtetramethylrhodamine analogs. The coumarin scaffold was explored first,synthesizing the azetidinyl coumarins 39 and 40 and the pyrrolidinylpair 41 and 42 (FIG. 43). Consistent with the other dyes, the spectralproperties of the deuterated azetidine compound 40 was similar to theparent 39 with similar ε and a small decrease in Φ. For the pyrrolidinylcompounds the differences were more pronounced, with deuterated dye 42showing significantly higher Φ compared to 41; the ε for the two dyeswas equivalent (FIG. 43). We also synthesized the pyrrolidinyl oxazinecompound 43 and deuterated congener 44. Like the otherpyrrolidine-containing dyes, the deuterium substitution caused asubstantial increase in Φ.

In conclusion, deuteration of the N-alkyl groups of rhodamine dyeselicits substantial improvements in performance. For standard tetraalkyldyes 1, 14, 17-22, increases in both extinction coefficient and quantumyields (FIG. 6, FIG. 10) are seen. For the already optimized azetidinecontaining dyes (15-16), deuteration did not further improve brightnessin vitro, but it did result in suppressed ¹O₂ generation (FIG. 17) andslower dealkylation (FIG. 18, FIG. 19) as well as improved brightnessand photostability as the HaloTag conjugates (FIG. 21, FIG. 22). Inaddition, the deuterated pyrrolidinyl compound 18 showed equivalentbrightness to the azetidinyl dyes in vitro (FIG. 6, FIG. 30) and itsderivatives exhibited superior brightness and photostability insidecells (FIG. 41, FIG. 42). These improvements due to deuteriumsubstitution were generalizable to carborhodamines (e.g., 29, FIG. 30),coumarins (e.g., 42, FIG. 43), oxazines (e.g., 44, FIG. 43). Overall,this represents a new strategy for improving the performance offluorophores, especially in the live-cell environment.

References: (1) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. Bright ideasfor chemical biology. 2008, 3, 142-155. (2) Beija, M.; Afonso, C. A. M.;Martinho, J. M. G. Chem. Soc. Rev. Synthesis and applications ofrhodamine derivatives as fluorescent probes. 2009, 38, 2410-2433. (3)Lavis, L. D.; Raines, R. T. ACS Chem. Biol. Bright building blocks forchemical biology. 2014, 9, 855-866. (4) Zheng, Q.; Lavis, L. D. Curr.Opin. Chem. Biol. Development of photostable fluorophores for molecularimaging. 2017, 39, 32-38. (5) Grimm, J. B.; English, B. P.; Chen, J.;Slaughter, J. P.; Zhang, Z.; Revyakin, A.; Patel, R.; Macklin, J. J.;Normanno, D.; Singer, R. H.; Lionnet, T.; Lavis, L. D. Nat. Methods Ageneral method to improve fluorophores for live-cell and single-moleculemicroscopy. 2015, 12, 244-250. (6) Hull, L. A.; Davis, G. T.;Rosenblatt, D. H.; Williams, H. K. R.; Weglein, R. C. J. Am. Chem. Soc.Oxidations of Amines. III. Duality of Mechanism in the Reaction ofAmines with Chlorine Dioxide. 1967, 89, 1163-1170. (7) Turro, N. J.;Ramamurthy, V.; Scaiano, J. C. Modern Molecular Photochemistry ofOrganic Molecules; University Science Books, 2010. (8) Dawson, W. R.;Windsor, M. W. J. Phys. Chem. Fluorescence yields of aromatic compounds.1968, 72, 3251-3260. (9) Kolmakov, K.; Belov, V. N.; Bierwagen, J.;Ringemann, C.; Muller, V.; Eggeling, C.; Hell, S. W. Chem. Eur. J.Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy.2010, 16, 158-166. (10) Frampton, M. J.; Accorsi, G.; Armaroli, N.;Rogers, J. E.; Fleitz, P. A.; McEwan, K. J.; Anderson, H. L. Org.Biomol. Chem. Synthesis and near-infrared luminescence of a deuteratedconjugated porphyrin dimer for probing the mechanism of non-radiativedeactivation. 2007, 5, 1056-1061. (11) Clemen, M.; Gernert, C.; Peters,J.; Grotemeyer, J. Eur. J. Mass Spectrom. Fragmentation reactions oflabeled and unlabeled rhodamine B in a high-resolution Fourier transformion cyclotron resonance mass spectrometer. 2013, 19, 135-139. (12)Peters, J.; Clemen, M.; Grotemeyer, J. Anal. Bioanal. Chem.Fragmentation of deuterated rhodamine B derivates by laser andcollisional activation in an FT-ICR mass spectrometer. 2013, 405,7061-9. (13) Pal, H.; Nagasawa, Y.; Tominaga, K.; Yoshihara, K. J. Phys.Chem. Deuterium isotope effect on ultrafast intermolecular electrontransfer. 1996, 100, 11964-11974. (14) Grimm, J. B.; Lavis, L. D. Org.Lett. Synthesis of rhodamines from fluoresceins using Pd-catalyzed C—Ncross-coupling. 2011, 13, 6354-7. (15) Los, G. V.; Encell, L. P.;McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M.G.; Learish, R.; Ohana, R. F.; Urh, M. ACS Chem. Biol. HaloTag: A novelprotein labeling technology for cell imaging and protein analysis. 2008,3, 373-382. (16) Liu, Z.; Legant, W. R.; Chen, B. C.; Li, L.; Grimm, J.B.; Lavis, L. D.; Betzig, E.; Tjian, R. Elife 3D imaging of Sox2enhancer clusters in embryonic stem cells. 2014, 3, e04236. (17) Grimm,J. B.; Sung, A. J.; Legant, W. R.; Hulamm, P.; Matlosz, S. M.; Betzig,E.; Lavis, L. D. ACS Chem. Biol. Carbofluoresceins and carborhodaminesas scaffolds for high-contrast fluorogenic probes. 2013, 8, 1303-1310.

“Alkyl” refers to an alkane missing one hydrogen and having the generalformula C_(n)H_(2n+1). Examples of lower alkyls (C1-C5) include: methyl;ethyl; propyl; butyl; and pentyl. Other, nonlimiting examples of alkylsare: hexyl; heptyl; octyl; nonyl; and decyl.

“Deuterated”, as in “deuterated compound, refers to a synthesizedcompound that has significantly more deuterium included than would bepredicted by natural isotopic abundance. Typically, when “D”,designating deuterium, is used instead of “H”, designating hydrogen thatis more than 98% hydrogen-1, in a chemical structure, it refers tohydrogen that is more than 50% deuterium. In certain cases, it refers tohydrogen that is more than 60%, more than 70%, more than 80%, more than90%, more than 95%, more than 97.5%, more than 98.0% or more than 98.5%deuterium.

“Substituted alkyl” refers to an alkyl where one or more hydrogen atomshave been replaced with a different substituent. Nonlimiting examples ofsuch substituents include: alkyl; alkenyl; alkynyl; cycloalkyl;cycloalkenyl; heterocycloalkyl; heterocycloalkenyl; aromatic group;heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH; CN; NO₂; CF₃;C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Aryl” refers to a cyclic or multi-cyclic, planar molecule with a ringof resonance bonds that exhibit more stability than other geometric orconnective arrangements with the same set of atoms. Nonlimiting examplesof aromatic groups include: phenyl; naphthyl; anthracenyl; andphenanthrenyl.

“Substituted aryl” refers to an aromatic group where one or morehydrogen atoms have been replaced with a different substituent.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Cycloalkyl” refers to a cycloalkane missing one hydrogen and having thegeneral formula C_(n)H_(2n+1). Nonlimiting examples of cycloalkylsinclude: cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl; cycloheptyl;cyclooctyl; cyclononyl; and cyclodecyl.

“Substituted cycloalkyl” refers to a cycloalkyl where one or morehydrogen atoms have been replaced with a different substituent.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“HaloTag” refers to a protein tag including a modified haloalkanedehalogenase designed to covalently bind to synthetic ligands. Thesynthetic ligands comprise a chloroalkane linker attached to a varietyof molecules. Nonlimiting examples of such molecules include: biotin;fluorescent dyes (e.g., Coumarin, Oregon Green, Alexa Fluor 488, diAcFAMand TMR); affinity handles; and solid surfaces. See, for example, Los etal., “A Novel Protein Labeling Technology for Cell Imaging and ProteinAnalysis”, ACS Chem. Biol. 2008, 3, 373-382, which isincorporated-by-reference into this document for all purposes.

The present invention provides deuterated fluorophores. FIG. 44 shows ageneral structure for such a deuterated fluorophore, where thesubstituents of the compound are as follows: R₁ is independentlyselected from halogen, H, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂,CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl), COO(aryl),C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl and substituted alkyl,aryl and substituted aryl, alkenyl and substituted alkenyl or where theR₁ and R₁′ substituents, taken together with the carbon atoms to whichthey are bonded, form a substituted or unsubstituted cycloalkyl ringcontaining 3, 4, 5, 6, 7, 8, or 9 carbon atoms; R₁′ is independentlyselected from halogen, H, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂,CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl), COO(aryl),C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl and substituted alkyl,aryl and substituted aryl, alkenyl and substituted alkenyl and only oneof R₁ and R₁′ can be D when X is CF₃, and only one of R₁ and R₁′ can beCD₃ when Q is O; R₂, R₃ and R₄ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), NH₂,NH(alkyl), N(alkyl)₂, N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂, CHO,C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl),C(O)NH(aryl), PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl andsubstituted aryl, alkenyl and substituted alkenyl; Q is selected fromC(alkyl)₂, NH, N(alkyl), O, S, Si(alkyl)₂, SO₂, P(O)(alkyl) P(O)(aryl)and Se; W is selected from C and N; X is selected from H, alkyl,substituted alkyl (e.g., CF₃), aryl, substituted aryl, halogen, CN, OH,O(alkyl), SH, S(alkyl), S(aryl), NH₂, NH₂, NH(alkyl), N(alkyl)₂,N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂, CHO, COOH, COO(alkyl), COO(aryl),PO₃H₂ and SO₃H; Y is selected from H, C(alkyl)₂, NH₂, N(alkyl),N(alkyl)₂, NH(aryl), NH(aryl)₂, O and S; Z is selected from H, halogen,CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl),N(alkyl)₂, N(alkyl)₃, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl, and substituted alkyl or Z and Y, taken togetherwith the carbon atoms to which they are bonded, form a substituted orunsubstituted cycloalkyl ring containing 4, 5, 6, 7 or 8 ring carbonatoms, or Z and Y, taken together with the carbon atoms to which theyare bonded, form a substituted of unsubstituted aryl ring.

FIG. 45 shows four structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃ andR₄ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃,NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH,COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyland substituted alkyl, aryl and substituted aryl, alkenyl andsubstituted alkenyl; W is selected from C and N; X is selected from H,alkyl, substituted alkyl, aryl, substituted aryl, halogen, CN, OH,O(alkyl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃,NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH,COO(alkyl), COO(aryl), PO₃H₂ and SO₃H, but X is not CF₃; Z is selectedfrom H, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl, and substituted alkyl.

FIG. 46 shows four structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃ andR₄ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, N(alkyl)₃,NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH,COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyland substituted alkyl, aryl and substituted aryl, alkenyl andsubstituted alkenyl; W is selected from C and N; X is selected from H,alkyl, substituted alkyl (e.g., CF₃), aryl, substituted aryl, halogen,CN, OH, O(alkyl), SH, S(alkyl), S(aryl), amine, NO₂, CHO, COOH,COO(alkyl), COO(aryl), PO₃H₂ and SO₃H; Z is selected from H, halogen,CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl),N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH,COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyland substituted alkyl, aryl and substituted aryl, alkenyl andsubstituted alkenyl.

FIG. 47 shows four structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃ andR₄ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, C(O)(alkyl), C(O)(aryl), CHO, COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl; W is selected from C and N; X is selected from H, alkyl,substituted alkyl (e.g., CF₃), aryl, substituted aryl, halogen, CN, OH,O(alkyl), SH, S(alkyl), S(aryl), amine, NO₂, CHO, COOH, COO(alkyl),COO(aryl), PO₃H₂ and SO₃H; NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl.

FIG. 48 shows four structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃ andR₄ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl; W is selected from C and N; X is selected from H, alkyl,substituted alkyl (e.g., CF₃), aryl, substituted aryl, halogen, CN, OH,O(alkyl), SH, S(alkyl), S(aryl), amine, NO₂, CHO, COOH, COO(alkyl),COO(aryl), PO₃H₂ and SO₃H; Z is selected from H, halogen, CN, OH,O(alkyl), O(aryl), SH, S(alkyl), S(aryl), NH₂, NH(alkyl), N(alkyl)₂,NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH,COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyland substituted alkyl, aryl and substituted aryl, alkenyl andsubstituted alkenyl.

FIG. 49 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl.

FIG. 50 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl.

FIG. 51 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl.

FIG. 52 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl; R₁₂ and R₁₃ are independently selected from CH₃, CH₂CH₃,CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

FIG. 53 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl.

FIG. 54 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl.

FIG. 55 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R¹⁰ and R¹¹ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl; R₁₂ and R₁₃ are independently selectedfrom CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

FIG. 56 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl; R₁₂ and R₁₃ are independently selected from CH₃, CH₂CH₃,CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

FIG. 57 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl.

FIG. 58 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl.

FIG. 59 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl.

FIG. 60 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl; R₁₂ and R₁₃ are independently selectedfrom CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

FIG. 61 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl; R₁₂ and R₁₃ are independently selected from CH₃, CH₂CH₃,CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

FIG. 62 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl.

FIG. 63 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl.

FIG. 64 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl; R₁₂ and R₁₃ are independently selectedfrom CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

FIG. 65 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₅, R₆, R₇and R₈ are independently selected from H, halogen, D, CN, OH, O(alkyl),O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂, NH(alkyl), N(alkyl)₂, NH(aryl),NH(aryl)₂, NO₂, CHO, C(O)(alkyl), C(O)(aryl), COOH, COO(alkyl),COO(aryl), C(O)NH(alkyl), C(O)NH(aryl), PO₃H₂, SO₃H, alkyl andsubstituted alkyl, aryl and substituted aryl, alkenyl and substitutedalkenyl; R₁₂ and R₁₃ are independently selected from CH₃, CH₂CH₃,CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

FIG. 66 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl; R₁₂ and R₁₃ are independently selectedfrom CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

FIG. 67 shows two structures for deuterated fluorophores according tothe present invention. Substituents for those structures are: R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from H,halogen, D, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), N₃, NH₂,NH(alkyl), N(alkyl)₂, NH(aryl), NH(aryl)₂, NO₂, CHO, C(O)(alkyl),C(O)(aryl), COOH, COO(alkyl), COO(aryl), C(O)NH(alkyl), C(O)NH(aryl),PO₃H₂, SO₃H, alkyl and substituted alkyl, aryl and substituted aryl,alkenyl and substituted alkenyl; R₁₂ and R₁₃ are independently selectedfrom CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, aryl and substituted aryl.

The synthesis of hydrogen-1 analogs of rhodamine and coumarinderivatives has been reported. See, for example, Beija, M.; Alfonso, C.A. M.; Martinho, J. M. G. Chem. Soc. Rev. Synthesis and applications ofrhodamine derivatives as fluorescent probes. 2009, 38, 2410-2433(rhodamine derivatives) and Vekariya, R; Patel, H. Syn. Comm. Recentadvances in the synthesis of coumarin derivatives via KnoevenagelCondensation: A review. 2014, 44, 2756-2788 (coumarin derivatives).

Deuterated fluorophores according to the present invention can besynthesized using any suitable method. One synthetic method involves across-coupling approach. See, for example, Grimm, J. B.; Lavis, L. D.Org. Lett. Synthesis of rhodamines from fluoresceins using Pd-catalyzedC—N cross-coupling. 2011, 13, 6354-6357. Deuterated cross-couplingcompounds such as pyrrolidine-d₈ and piperidine-d₁₁ can be purchased,e.g., Sigma-Aldrich, or synthesized using suitable methods, e.g.Atzrodt, J.; Derdau, V.; Holla, W.; Beller, M.; Neubert, L.; Michalik,D. European Patent Application EP2714624A1. Process for the preparationof deuterated compounds containing n-alkyl groups. 2012, and referencestherein. FIGS. 68-70 show schemes for synthesizing compounds of thepresent invention.

Deuterated fluorophores according to the present invention can be usedfor any suitable purpose. Nonlimiting examples of such use include useas/for: a dye; fluorescence microscopy, flow cytometry, fluorescencecorrelation spectroscopy and ELISA.

EXPERIMENTALS Example 1.2-(3,6-Bis(bis(methyl-d₃)amino)xanthylium-9-yl)benzoate

A vial was charged with fluorescein ditriflate (Grimm, J. B.; Lavis, L.D. Org. Lett. 2011, 13, 6354-6357; 150 mg, 0.251 mmol),dimethyl-d₆-amine hydrochloride (52.9 mg, 0.604 mmol, 2.4 eq), Pd₂dba₃(23.0 mg, 25.1 μmol, 0.1 eq), XPhos (36.0 mg, 75.4 μmol, 0.3 eq), andCs₂CO₃ (393 mg, 1.21 mmol, 4.8 eq). The vial was sealed andevacuated/backfilled with nitrogen (3×). Dioxane (1.5 mL) was added, andthe reaction was flushed again with nitrogen (3×). The reaction was thenstirred at 100° C. for 4 h. It was subsequently cooled to roomtemperature, diluted with MeOH, deposited onto Celite, and concentratedto dryness. Purification by silica gel chromatography (0-10% MeOH (2 MNH₃)/CH₂Cl₂, linear gradient; dry load on Celite) followed by reversephase HPLC (10-50% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive) afforded the title compound (92 mg, 71%, TFA salt) as a darkred solid. ¹H NMR (CD₃OD, 400 MHz) δ 8.37-8.32 (m, 1H), 7.86 (td, J=7.5,1.5 Hz, 1H), 7.80 (td, J=7.6, 1.5 Hz, 1H), 7.44-7.38 (m, 1H), 7.15 (d,J=9.5 Hz, 2H), 7.05 (dd, J=9.5, 2.5 Hz, 2H), 6.97 (d, J=2.5 Hz, 2H);Analytical HPLC: t_(R)=10.5 min, >99% purity (5 μL injection; 10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 550 nm); HRMS(ESI) calcd for C₂₄H₁₁D₁₂N₂O₃ [M+H]⁺399.2456, found 399.2454.

Example 2. 2-(3,6-Bis(azetidin-1-yl-d₆)xanthylium-9-yl)benzoate

The title compound (81%, dark red solid) was prepared from fluoresceinditriflate and azetidine-2,2,3,3,4,4-d₆ hydrochloride (Helal, C. J.;Chappie, T. A.; Humphrey, J. M. Int. Pat. Appl. WO 2012/168817 A1, Dec.13, 2012) according to the procedure described for Example 1. ¹H NMR(CD₃OD, 400 MHz) δ 8.09-8.06 (m, 1H), 7.64 (td, J=7.5, 1.6 Hz, 1H), 7.59(td, J=7.4, 1.6 Hz, 1H), 7.22-7.19 (m, 1H), 7.18 (d, J=9.2 Hz, 2H), 6.54(dd, J=9.2, 2.2 Hz, 2H), 6.46 (d, J=2.2 Hz, 2H); Analytical HPLC:t_(R)=11.3 min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd forC₂₆H₁₁D₁₂N₂O₃ [M+H]⁺ 423.2456, found 423.2454.

Example 3. 2-(3,6-Bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)benzoate

The title compound (76%, dark red-purple solid) was prepared fromfluorescein ditriflate and pyrrolidine-2,2,3,3,4,4,5,5-d₈ according tothe procedure described for Example 1. ¹H NMR (CD₃OD, 400 MHz) δ8.11-8.07 (m, 1H), 7.65 (td, J=7.5, 1.6 Hz, 1H), 7.60 (td, J=7.4, 1.6Hz, 1H), 7.26 (d, J=9.3 Hz, 2H), 7.25-7.22 (m, 1H), 6.85 (dd, J=9.3, 2.4Hz, 2H), 6.75 (d, J=2.3 Hz, 2H); Analytical HPLC: t_(R)=12.4 min, XX %purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 550 nm); HRMS (ESI) calcd for C₂₈H₁₁D₁₆N₂O₃[M+H]⁺455.3020, found 455.3018.

Example 4. 2-(3,6-Bis(piperidin-1-yl-d₁₀)xanthylium-9-yl)benzoate

The title compound (96%, dark red-purple solid) was prepared fromfluorescein ditriflate and piperidine-d₁₁ according to the proceduredescribed for Example 1. ¹H NMR (CD₃OD, 400 MHz) δ 8.11-8.05 (m, 1H),7.66 (td, J=7.4, 1.8 Hz, 1H), 7.62 (td, J=7.3, 1.7 Hz, 1H), 7.26-7.21(m, 1H), 7.17 (d, J=9.4 Hz, 2H), 7.06 (dd, J=9.4, 2.6 Hz, 2H), 7.01 (d,J=2.5 Hz, 2H); Analytical HPLC: t_(R)=12.7 min, >99% purity (5 μLinjection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 20 min run; 1 mL/min flow; ESI; positive ion mode; detectionat 550 nm); HRMS (ESI) calcd for C₃₀H₁₁D₂₀N₂O₃ [M+H]⁺487.3585, found487.3588.

Example 5. 2-(3,6-Bis(morpholino-d₈)xanthylium-9-yl)benzoate

The title compound (88%, pink solid) was prepared from fluoresceinditriflate and morpholine-2,2,3,3,5,5,6,6-d₈ according to the proceduredescribed for Example 1. ¹H NMR (CD₃OD, 400 MHz) δ 8.03-7.99 (m, 1H),7.74 (td, J=7.4, 1.4 Hz, 1H), 7.69 (td, J=7.4, 1.2 Hz, 1H), 7.21-7.17(m, 1H), 6.83 (d, J=2.4 Hz, 2H), 6.77 (dd, J=8.9, 2.5 Hz, 2H), 6.71 (d,J=8.9 Hz, 2H); Analytical HPLC: t_(R)=10.2 min, >99% purity (5 μLinjection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 20 min run; 1 mL/min flow; ESI; positive ion mode; detectionat 550 nm); HRMS (ESI) calcd for C₂₈H₁₁D₁₆N₂O₅ [M+H]⁺487.2919, found487.2926.

Example 6.2-(3,7-Bis(bis(methyl-d₃)amino)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (89%, off-white solid) was prepared fromSi-fluorescein ditriflate (Grimm, J. B. et al. Nat. Methods 2015, 12,244-250) and dimethyl-d₆-amine hydrochloride according to the proceduredescribed for Example 1. ¹H NMR (CDCl₃, 400 MHz) δ 7.96 (dt, J=7.6, 1.0Hz, 1H), 7.63 (td, J=7.5, 1.2 Hz, 1H), 7.53 (td, J=7.5, 1.0 Hz, 1H),7.29 (dt, J=7.7, 0.9 Hz, 1H), 6.96 (d, J=2.9 Hz, 2H), 6.78 (d, J=8.9 Hz,2H), 6.54 (dd, J=8.9, 2.9 Hz, 2H), 0.64 (s, 3H), 0.60 (s, 3H);Analytical HPLC: t_(R)=10.4 min, >99% purity (5 μL injection; 10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 650 nm); MS(ESI) calcd for C₂₆H₁₇D₁₂N₂O₂Si [M+H]⁺441.3, found 441.2.

Example 7.2-(3,7-Bis(azetidin-1-yl-d₆)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (57%, off-white solid) was prepared fromSi-fluorescein ditriflate and azetidine-2,2,3,3,4,4-d₆ hydrochlorideaccording to the procedure described for Example 1. ¹H NMR (CDCl₃, 400MHz) δ 7.96 (dt, J=7.6, 1.0 Hz, 1H), 7.64 (td, J=7.5, 1.1 Hz, 1H), 7.54(td, J=7.5, 0.8 Hz, 1H), 7.31 (dt, J=7.7, 0.9 Hz, 1H), 6.75 (d, J=8.7Hz, 2H), 6.67 (d, J=2.6 Hz, 2H), 6.25 (dd, J=8.6, 2.7 Hz, 2H), 0.61 (s,3H), 0.59 (s, 3H); Analytical HPLC: t_(R)=12.5 min, >99% purity (5 μLinjection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 20 min run; 1 mL/min flow; ESI; positive ion mode; detectionat 650 nm); HRMS (ESI) calcd for C₂₈H₁₇D₁₂N₂O₂Si [M+H]⁺465.2746, found465.2749.

Example 8.2-(5,5-Dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (96%, pale blue solid) was prepared fromSi-fluorescein ditriflate and pyrrolidine according to the proceduredescribed for Example 1. ¹H NMR (CDCl₃, 400 MHz) δ 7.95 (dt, J=7.6, 0.9Hz, 1H), 7.62 (td, J=7.5, 1.1 Hz, 1H), 7.52 (td, J=7.5, 0.9 Hz, 1H),7.31-7.27 (m, 1H), 6.79 (d, J=2.7 Hz, 2H), 6.76 (d, J=8.8 Hz, 2H), 6.38(dd, J=8.8, 2.8 Hz, 2H), 3.34-3.24 (m, 8H), 2.05-1.93 (m, 8H), 0.63 (s,3H), 0.60 (s, 3H); ¹³C NMR (CDCl₃, 101 MHz) δ 171.0 (C), 154.9 (C),146.8 (C), 137.2 (C), 133.7 (CH), 131.0 (C), 128.6 (CH), 128.4 (CH),127.2 (C), 125.6 (CH), 124.7 (CH), 115.9 (CH), 112.7 (CH), 92.5 (C),47.6 (CH₂), 25.6 (CH₂), 0.6 (CH₃), −1.3 (CH₃); Analytical HPLC: >99%purity (5 μL injection; 30-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 650 nm); HRMS (ESI) calcd for C₃₀H₃₃N₂O₂Si[M+H]⁺481.2306, found 481.2317.

Example 9.2-(5,5-Dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (90%, off-white solid) was prepared fromSi-fluorescein ditriflate and pyrrolidine-2,2,3,3,4,4,5,5-d₈ accordingto the procedure described for Example 1. ¹H NMR (CDCl₃, 400 MHz) δ 7.95(dt, J=7.7, 1.0 Hz, 1H), 7.61 (td, J=7.5, 1.1 Hz, 1H), 7.52 (td, J=7.5,0.9 Hz, 1H), 7.28 (dt, J=7.8, 1.0 Hz, 1H), 6.79 (d, J=2.8 Hz, 2H), 6.76(d, J=8.8 Hz, 2H), 6.38 (dd, J=8.8, 2.8 Hz, 2H), 0.63 (s, 3H), 0.59 (s,3H); Analytical HPLC: t_(R)=13.0 min, >99% purity (5 μL injection;10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive;20 min run; 1 mL/min flow; ESI; positive ion mode; detection at 650 nm);HRMS (ESI) calcd for C₃₀H₁₇D₁₆N₂O₂Si [M+H]⁺497.3310, found 497.3312.

Example 10.2-(3,6-Bis(bis(methyl-d₃)amino)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)benzoate

The title compound (90%, pale blue solid) was prepared fromcarbofluorescein ditriflate (Grimm, J. B. et al. ACS Chem. Biol. 2013,8, 1303-1310) and dimethyl-d₆-amine hydrochloride according to theprocedure described for Example 1. ¹H NMR (CDCl₃, 400 MHz) δ 8.01-7.96(m, 1H), 7.58 (td, J=7.4, 1.5 Hz, 1H), 7.53 (td, J=7.4, 1.3 Hz, 1H),7.09-7.04 (m, 1H), 6.88 (d, J=2.7 Hz, 2H), 6.60 (d, J=8.8 Hz, 2H), 6.50(dd, J=8.8, 2.6 Hz, 2H), 1.88 (s, 3H), 1.77 (s, 3H); Analytical HPLC:t_(R)=10.3 min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; detection at 600 nm); MS (ESI) calcd forC₂₇H₁₇D₁₂N₂O₂ [M+H]⁺425.3, found 425.2.

Example 11.2-(3,6-Bis(azetidin-1-yl-d₆)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)benzoate

A vial was charged with carbofluorescein ditriflate (150 mg, 0.241mmol), azetidine-2,2,3,3,4,4-d₆ hydrochloride (120 mg, 1.20 mmol, 5 eq),RuPhos-G3-palladacycle (20.2 mg, 24.1 μmol, 0.1 eq), RuPhos (11.2 mg,24.1 μmol, 0.1 eq), and Cs₂CO₃ (628 mg, 1.93 mmol, 8 eq). The vial wassealed and evacuated/backfilled with nitrogen (3×). Dioxane (2 mL) wasadded, and the reaction was flushed again with nitrogen (3×). Thereaction was then stirred at 100° C. for 4 h. It was subsequently cooledto room temperature, filtered through Celite with CH₂Cl₂, andconcentrated to dryness. Purification by silica gel chromatography(10-100% EtOAc/hexanes, linear gradient afforded the title compound (59mg, 55%) as a pale blue solid. ¹H NMR (CDCl₃, 400 MHz) δ 8.00-7.95 (m,1H), 7.58 (td, J=7.4, 1.4 Hz, 1H), 7.53 (td, J=7.4, 1.2 Hz, 1H),7.08-7.04 (m, 1H), 6.58 (d, J=2.4 Hz, 2H), 6.55 (d, J=8.6 Hz, 2H), 6.20(dd, J=8.6, 2.4 Hz, 2H), 1.82 (s, 3H), 1.72 (s, 3H); Analytical HPLC:t_(R)=11.8 min, 98.5% purity (5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; detection at 600 nm); HRMS (ESI) calcd forC₂₉H₁₇D₁₂N₂O₂ [M+H]⁺449.2977, found 449.2980.

Example 12.2-(10,10-Dimethyl-3,6-bis(pyrrolidin-1-yl-d₈)anthracen-9-ylium-9(10H)-yl)benzoate

The title compound (91%, pale blue solid) was prepared fromcarbofluorescein ditriflate and pyrrolidine-2,2,3,3,4,4,5,5-d₈ accordingto the procedure described for Example 1. ¹H NMR (CDCl₃, 400 MHz) δ8.00-7.96 (m, 1H), 7.57 (td, J=7.3, 1.5 Hz, 1H), 7.52 (td, J=7.4, 1.3Hz, 1H), 7.08-7.04 (m, 1H), 6.72 (d, J=2.5 Hz, 2H), 6.58 (d, J=8.7 Hz,2H), 6.35 (dd, J=8.7, 2.5 Hz, 2H), 1.88 (s, 3H), 1.77 (s, 3H);Analytical HPLC: t_(R)=9.8 min, >99% purity (5 μL injection; 30-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 600 nm); HRMS(ESI) calcd for C₃₁H₁₇D₁₆N₂O₂ [M+H]⁺481.3541, found 481.3545.

Example 13.2-(3,6-Bis(azetidin-1-yl-d₆)xanthylium-9-yl)-4-(tert-butoxycarbonyl)benzoate

The title compound (72%, dark red-purple solid) was prepared from6-tert-butoxycarbonylfluorescein ditriflate (Grimm, J. B. et al. Nat.Methods 2015, 12, 244-250) and azetidine-2,2,3,3,4,4-d₆ hydrochlorideaccording to procedure described for Example 1. ¹H NMR (CDCl₃, 400 MHz)δ 8.19 (dd, J=8.0, 1.3 Hz, 1H), 8.01 (dd, J=8.0, 0.5 Hz, 1H), 7.77-7.70(m, 1H), 6.53 (d, J=8.6 Hz, 2H), 6.21 (d, J=2.3 Hz, 2H), 6.09 (dd,J=8.6, 2.3 Hz, 2H), 1.54 (s, 9H); Analytical HPLC: t_(R)=12.9 min, >99%purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 550 nm); HRMS (ESI) calcd for C₃₁H₁₉D₁₂N₂O₅[M+H]⁺523.2981, found 523.2982.

Example 14.4-(tert-Butoxycarbonyl)-2-(3,6-di(pyrrolidin-1-yl)xanthylium-9-yl)benzoate

The title compound (59%, dark red-purple solid) was prepared from6-tert-butoxycarbonylfluorescein ditriflate and pyrrolidine according tothe procedure described for Example 1. ¹H NMR (CD₃OD, 400 MHz) δ 8.20(dd, J=8.1, 1.7 Hz, 1H), 8.11 (d, J=8.1 Hz, 1H), 7.78 (d, J=1.7 Hz, 1H),7.22 (d, J=9.4 Hz, 2H), 6.87 (dd, J=9.4, 2.3 Hz, 2H), 6.77 (d, J=2.3 Hz,2H), 3.65-3.52 (m, 8H), 2.20-2.06 (m, 8H), 1.59 (s, 9H); ¹³C NMR (CD₃OD,101 MHz) δ 172.4 (C), 166.1 (C), 162.2 (C), 158.9 (C), 156.1 (C), 145.9(C), 133.8 (C), 133.6 (C), 132.7 (CH), 131.5 (CH), 131.2 (CH), 131.0(CH), 115.9 (CH), 115.1 (C), 97.6 (CH), 83.1 (C), 49.9 (CH₂), 28.4(CH₃), 26.2 (CH₂); Analytical HPLC: t_(R)=13.7 min, >99% purity (5 μLinjection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 20 min run; 1 mL/min flow; ESI; positive ion mode; detectionat 550 nm); HRMS (ESI) calcd for C₃₃H₃₅N₂O₅ [M+H]⁺539.2540, found539.2544.

Example 15.2-(3,6-Bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)-4-(tert-butoxycarbonyl)benzoate

The title compound (58%, dark red-purple solid) was prepared from6-tert-butoxycarbonylfluorescein ditriflate andpyrrolidine-2,2,3,3,4,4,5,5-d₈ according to the procedure described forExample 1. ¹H NMR (CD₃OD, 400 MHz) δ 8.20 (dd, J=8.2, 1.7 Hz, 1H), 8.11(d, J=8.1 Hz, 1H), 7.78 (d, J=1.6 Hz, 1H), 7.21 (d, J=9.3 Hz, 2H), 6.87(dd, J=9.3, 2.4 Hz, 2H), 6.76 (d, J=2.3 Hz, 2H), 1.59 (s, 9H);Analytical HPLC: t_(R)=13.6 min, >99% purity (5 μL injection; 10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 550 nm); HRMS(ESI) calcd for C₃₃H₁₉D₁₆N₂O₅ [M+H]⁺555.3545, found 555.3544.

Example 16.2-(3,7-Bis(azetidin-1-yl-d₆)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(tert-butoxycarbonyl)benzoate

The title compound (48%, off-white foam) was prepared from6-tert-butoxycarbonyl-Si-fluorescein ditriflate (Grimm, J. B. et al.Nat. Methods 2015, 12, 244-250) and azetidine-2,2,3,3,4,4-d₆hydrochloride according to the procedure described for Example 1. ¹H NMR(CDCl₃, 400 MHz) δ 8.11 (dd, J=8.0, 1.3 Hz, 1H), 7.95 (dd, J=8.0, 0.6Hz, 1H), 7.83-7.80 (m, 1H), 6.82 (d, J=8.7 Hz, 2H), 6.66 (d, J=2.7 Hz,2H), 6.29 (dd, J=8.7, 2.7 Hz, 2H), 1.54 (s, 9H), 0.64 (s, 3H), 0.58 (s,3H); Analytical HPLC: t_(R)=14.1 min, 98.8% purity (5 μL injection;10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive;20 min run; 1 mL/min flow; ESI; positive ion mode; detection at 650 nm);HRMS (ESI) calcd for C₃₃H₂₅D₁₂N₂O₄Si [M+H]⁺565.3270, found 565.3277.

Example 17.4-(tert-Butoxycarbonyl)-2-(5,5-dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (81%, off-white solid) was prepared from6-tert-butoxycarbonyl-Si-fluorescein ditriflate and pyrrolidineaccording to the procedure described for Example 1. ¹H NMR (CDCl₃, 400MHz) δ 8.10 (dd, J=8.0, 1.3 Hz, 1H), 7.96 (dd, J=8.0, 0.5 Hz, 1H),7.83-7.79 (m, 1H), 6.84 (d, J=8.8 Hz, 2H), 6.79 (d, J=2.7 Hz, 2H), 6.44(dd, J=8.8, 2.8 Hz, 2H), 3.35-3.25 (m, 8H), 2.04-1.95 (m, 8H), 1.53 (s,9H), 0.67 (s, 3H), 0.60 (s, 3H); ¹³C NMR (CDCl₃, 101 MHz) δ 170.6 (C),164.5 (C), 155.9 (C), 146.8 (C), 137.2 (C), 136.3 (C), 130.5 (C), 129.8(CH), 129.3 (C), 128.1 (CH), 125.5 (CH), 125.1 (CH), 115.9 (CH), 113.2(CH), 92.2 (C), 82.2 (C), 47.6 (CH₂), 28.2 (CH₃), 25.6 (CH₂), 0.2 (CH₃),−0.5 (CH₃); Analytical HPLC: t_(R)=14.6 min, >99% purity (5 μLinjection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 20 min run; 1 mL/min flow; ESI; positive ion mode; detectionat 650 nm); HRMS (ESI) calcd for C₃₅H₄₁N₂O₄Si [M+H]⁺581.2830, found581.2839.

Example 18.4-(tert-Butoxycarbonyl)-2-(5,5-dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (87%, off-white solid) was prepared from6-tert-butoxycarbonyl-Si-fluorescein ditriflate andpyrrolidine-2,2,3,3,4,4,5,5-d₈ according to the procedure described forExample 1. ¹H NMR (CDCl₃, 400 MHz) δ 8.10 (dd, J=8.0, 1.3 Hz, 1H), 7.95(dd, J=8.0, 0.6 Hz, 1H), 7.83-7.78 (m, 1H), 6.84 (d, J=8.8 Hz, 2H), 6.79(d, J=2.7 Hz, 2H), 6.43 (dd, J=8.8, 2.8 Hz, 2H), 1.53 (s, 9H), 0.67 (s,3H), 0.59 (s, 3H); Analytical HPLC: t_(R)=14.4 min, >99% purity (5 μLinjection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 20 min run; 1 mL/min flow; ESI; positive ion mode; detectionat 650 nm); HRMS (ESI) calcd for C₃₅H₂₅D₁₆N₂O₄Si [M+H]⁺597.3834, found597.3835.

Example 19.2-(3,6-Bis(azetidin-1-yl-d₆)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-(tert-butoxycarbonyl)benzoate

The title compound (42%, blue solid) was prepared from6-tert-butoxycarbonyl-carbofluorescein ditriflate (Grimm, J. B. et al.Nat. Methods 2017, 14, 987-994) and azetidine-2,2,3,3,4,4-d₆hydrochloride according to the procedure described for Example 11. ¹HNMR (CD₃OD, 400 MHz) δ 8.17 (dd, J=8.1, 1.5 Hz, 1H), 8.04 (d, J=8.1 Hz,1H), 7.53 (d, J=1.3 Hz, 1H), 6.72 (d, J=2.3 Hz, 2H), 6.67 (d, J=8.7 Hz,2H), 6.32 (dd, J=8.6, 2.3 Hz, 2H), 1.82 (s, 3H), 1.72 (s, 3H), 1.55 (s,9H); HRMS (ESI) calcd for C₃₄H₂₅D₁₂N₂O₄ [M+H]⁺549.3501, found 549.3503.

Example 20.4-(tert-Butoxycarbonyl)-2-(10,10-dimethyl-3,6-di(pyrrolidin-1-yl)anthracen-9-ylium-9(10H)-yl)benzoate

The title compound (79%, blue solid) was prepared from6-tert-butoxycarbonyl-carbofluorescein ditriflate and pyrrolidineaccording to the procedure described for Example 1. ¹H NMR (CD₃OD, 400MHz) δ 8.15 (dd, J=8.1, 1.7 Hz, 1H), 8.06 (d, J=8.1 Hz, 1H), 7.63 (d,J=1.6 Hz, 1H), 7.01 (d, J=2.7 Hz, 2H), 6.99 (d, J=9.4 Hz, 2H), 6.60 (dd,J=9.2, 2.4 Hz, 2H), 3.63-3.52 (m, 8H), 2.16-2.06 (m, 8H), 1.85 (s, 3H),1.77 (s, 3H), 1.58 (s, 9H); Analytical HPLC: t_(R)=14.2 min, >99% purity(5 μL injection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1%v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ion mode;detection at 600 nm); HRMS (ESI) calcd for C₃₆H₄₁N₂O₄ [M+H]⁺565.3061,found 565.3071.

Example 21.4-(tert-Butoxycarbonyl)-2-(10,10-dimethyl-3,6-bis(pyrrolidin-1-yl-d₈)anthracen-9-ylium-9(10H)-yl)benzoate

The title compound (50%, blue solid) was prepared from6-tert-butoxycarbonyl-carbofluorescein ditriflate andpyrrolidine-2,2,3,3,4,4,5,5-d₈ according to the procedure described forExample 1. ¹H NMR (CD₃OD, 400 MHz) δ 8.15 (dd, J=8.1, 1.6 Hz, 1H), 8.06(d, J=8.0 Hz, 1H), 7.64 (d, J=1.5 Hz, 1H), 7.01 (d, J=2.7 Hz, 2H), 7.00(d, J=9.0 Hz, 2H), 6.60 (dd, J=9.2, 2.4 Hz, 2H), 1.85 (s, 3H), 1.77 (s,3H), 1.58 (s, 9H); HRMS (ESI) calcd for C₃₆H₂₅D₁₆N₂O₄ [M+H]⁺581.4065,found 581.4064.

Example 22.2-(3,6-Di(azetidin-1-yl)xanthylium-9-yl-1,2,4,5,7,8-d₆)benzoate

Step 1: Phthalic anhydride (765 mg, 5.16 mol) and1,3-dihydroxybenzene-d₆ (1.20 g, 10.3 mmol, 2 eq) were combined inmethanesulfonic acid-d₄ (5 mL) and stirred at 85° C. for 48 h. The darkbrown reaction mixture was cooled to room temperature, poured into D₂O(40 mL), and vigorously stirred for 18 h. The resulting suspension wasfiltered; the filter cake was washed with D₂O and thoroughly dried toprovide 1.84 g crude fluorescein-1′,2′,4′,5′,7′,8′-d₆ as a brown solid.This material was suspended in CH₂Cl₂ (25 mL) and cooled to 0° C.Pyridine (3.50 mL, 43.2 mmol, 8 eq) and trifluoromethanesulfonicanhydride (3.64 mL, 21.6 mmol, 4 eq) were added, and the ice bath wasremoved. The reaction was stirred at room temperature for 18 h. It wassubsequently diluted with water and extracted with CH₂Cl₂ (2×). Thecombined organic extracts were washed with saturated CuSO₄ and brine,dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. Silicagel chromatography (0-30% EtOAc/hexanes, linear gradient) yielded 1.69 g(54%, 2 steps) of fluorescein-1′,2′,4′,5′,7′,8′-d₆ ditriflate as acolorless foam. ¹H NMR (CDCl₃, 400 MHz) δ 8.10-8.06 (m, 1H), 7.74 (td,J=7.4, 1.4 Hz, 1H), 7.70 (td, J=7.4, 1.2 Hz, 1H), 7.21-7.16 (m, 1H); ¹⁹FNMR (CDCl₃, 376 MHz) δ 73.13 (s); Analytical HPLC: t_(R)=15.3 min, >99%purity (5 μL injection; 30-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 254 nm); HRMS (ESI) calcd for C₂₂H₅D₆F₆O₉S₂[M+H]⁺603.0120, found 603.0127.

Step 2: The title compound (49%, purple solid) was prepared fromfluorescein-1′,2′,4′,5′,7′,8′-d₆ ditriflate (Step 1) and azetidineaccording to the procedure described for Example 1. ¹H NMR (CDCl₃, 400MHz) δ 8.02-7.95 (m, 1H), 7.63 (td, J=7.5, 1.3 Hz, 1H), 7.57 (td, J=7.4,1.1 Hz, 1H), 7.19-7.15 (m, 1H), 3.90 (t, J=7.3 Hz, 8H), 2.37 (p, J=7.3Hz, 4H); Analytical HPLC: t_(R)=10.8 min, 97.5% purity (5 μL injection;10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive;20 min run; 1 mL/min flow; ESI; positive ion mode; detection at 550 nm);HRMS (ESI) calcd for C₂₆H₁₇D₆N₂O₃ [M+H]⁺417.2080, found 417.2081.

Example 23.2-(3,6-Di(azetidin-1-yl)xanthylium-9-yl-1,2,4,5,7,8-d₆)benzoate-d₄

Step 1: The procedure described for Example 22, Step 1 was used toprepare fluorescein-1′,2′,4,4′,5,5′,6,7,7′,8′-d₁₀ ditriflate (50%, whitefoam) from 1,3-dihydroxybenzene-d₆ and phthalic anhydride-d₄. ¹⁹F NMR(CDCl₃, 376 MHz) δ 73.13 (s); Analytical HPLC: t_(R)=15.3 min, >99%purity (5 μL injection; 30-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 254 nm); HRMS (ESI) calcd for C₂₂HD₁₀F₆O₉S₂[M+H]⁺607.0371, found 607.0373.

Step 2: The title compound (74%, purple solid) was prepared fromfluorescein-1′,2′,4,4′,5,5′,6,7,7,′,8′-d₁₀ ditriflate (Step 1) andazetidine according to the procedure described for Example 1. ¹H NMR(CDCl₃, 400 MHz) δ 3.90 (t, J=7.3 Hz, 8H), 2.37 (p, J=7.3 Hz, 4H);Analytical HPLC: t_(R)=10.9 min, >99% purity (5 μL injection; 10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 550 nm); HRMS(ESI) calcd for C₂₆H₁₃D₁₀N₂O₃ [M+H]⁺421.2331, found 421.2322.

Example 24.2-(3,6-Bis(azetidin-1-yl-d₆)xanthylium-9-yl-1,2,4,5,7,8-d₆)benzoate

The title compound (90%, purple solid) was prepared fromfluorescein-1′,2′,4′,5′,7′,8′-d₆ ditriflate (Example 22, Step 1) andazetidine-2,2,3,3,4,4-d₆ hydrochloride according to the proceduredescribed for Example 1. ¹H NMR (CDCl₃, 400 MHz) δ 8.01-7.96 (m, 1H),7.63 (td, J=7.4, 1.3 Hz, 1H), 7.57 (td, J=7.4, 1.1 Hz, 1H), 7.19-7.14(m, 1H); Analytical HPLC: t_(R)=10.9 min, 98.4% purity (5 μL injection;10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive;20 min run; 1 mL/min flow; ESI; positive ion mode; detection at 550 nm);HRMS (ESI) calcd for C₂₆H₅D₁₈N₂O₃ [M+H]⁺429.2833, found 429.2834.

Example 25.2-(3,6-Bis(azetidin-1-yl-d₆)xanthylium-9-yl-1,2,4,5,7,8-d₆)benzoate-d₄

The title compound (64%, purple solid) was prepared fromfluorescein-1′,2′,4,4′,5,5′,6,7,7′,8′-d₁₀ ditriflate (Example 23,Step 1) and azetidine-2,2,3,3,4,4-d₆ hydrochloride according to theprocedure described for Example 1. Analytical HPLC: t_(R)=10.8 min, >99%purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 550 nm); HRMS (ESI) calcd for C₂₆HD₂₂N₂O₃[M+H]⁺433.3084, found 433.3083.

Example 26. 7-(Azetidin-1-yl-d₆)-4-methyl-2H-chromen-2-one

A vial was charged with 4-methylumbelliferone triflate (Kövér, J.;Antus, S. Z. Naturforsch., B: J Chem. Sci. 2005, 60, 792-796; 175 mg,0.568 mmol), azetidine-2,2,3,3,4,4-d₆ hydrochloride (141 mg, 1.42 mmol,2.5 eq), RuPhos-G3-palladacycle (23.7 mg, 28.4 μmol, 0.05 eq), RuPhos(13.2 mg, 28.4 μmol, 0.05 eq), and K₂CO₃ (314 mg, 2.27 mmol, 4 eq). Thevial was sealed and evacuated/backfilled with nitrogen (3×). Dioxane (3mL) was added, and the reaction was flushed again with nitrogen (3×).The reaction was then stirred at 100° C. for 18 h. It was subsequentlycooled to room temperature, deposited onto Celite, and concentrated todryness. Purification by silica gel chromatography (0-30% EtOAc/hexanes,linear gradient; dry load with Celite) afforded 20 mg (16%) of the titlecompound as a yellow solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.38 (d, J=8.6 Hz,1H), 6.30 (dd, J=8.6, 2.3 Hz, 1H), 6.22 (d, J=2.3 Hz, 1H), 5.97 (q,J=1.2 Hz, 1H), 2.34 (d, J=1.2 Hz, 3H); Analytical HPLC: t_(R)=11.9min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positiveion mode; detection at 350 nm); HRMS (ESI) calcd for C₁₃H₈D₆NO₂[M+H]⁺222.1396, found 222.1394.

Example 27. 4-Methyl-7-(pyrrolidin-1-yl)-2H-chromen-2-one

The title compound (92%, yellow solid) was prepared from4-methylumbelliferone triflate and pyrrolidine according to theprocedure described for Example 26. ¹H NMR (CDCl₃, 400 MHz) δ 7.39 (d,J=8.8 Hz, 1H), 6.48 (dd, J=8.8, 2.4 Hz, 1H), 6.38 (d, J=2.4 Hz, 1H),5.94 (q, J=1.1 Hz, 1H), 3.40-3.30 (m, 4H), 2.34 (d, J=1.1 Hz, 3H),2.10-1.99 (m, 4H); ¹³C NMR (CDCl₃, 101 MHz) δ 162.3 (C), 155.9 (C),153.2 (C), 150.5 (C), 125.5 (CH), 109.4 (C), 109.1 (CH), 108.8 (CH),98.0 (CH), 47.8 (CH₂), 25.6 (CH₂), 18.6 (CH₃); Analytical HPLC:t_(R)=13.2 min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; detection at 375 nm); HRMS (ESI) calcd forC₁₄H₁₆NO₂ [M+H]⁺230.1176, found 230.1180.

Example 28. 4-Methyl-7-(pyrrolidin-1-yl-d₈)-2H-chromen-2-one

The title compound (86%, yellow solid) was prepared from4-methylumbelliferone triflate and pyrrolidine-2,2,3,3,4,4,5,5-d₈according to the procedure described for Example 26. ¹H NMR (CDCl₃, 400MHz) δ 7.38 (d, J=8.8 Hz, 1H), 6.48 (dd, J=8.8, 2.4 Hz, 1H), 6.38 (d,J=2.4 Hz, 1H), 5.94 (q, J=1.1 Hz, 1H), 2.34 (d, J=1.1 Hz, 3H);Analytical HPLC: t_(R)=13.1 min, >99% purity (5 μL injection; 10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 375 nm); HRMS(ESI) calcd for C₁₄H₈D₈NO₂ [M+H]⁺238.1678, found 238.1682.

Example 29. 3,7-Di(pyrrolidin-1-yl)phenoxazin-5-ium trifluoroacetate

A vial was charged with 10-acetyl-10H-phenoxazine-3,7-diylbis(trifluoromethanesulfonate) (Grimm, J. B. et al. Nat. Methods 2015,12, 244-250; 250 mg, 0.480 mmol), Pd₂dba₃ (43.9 mg, 48.0 μmol, 0.1 eq),XPhos (68.6 mg, 0.144 mmol, 0.3 eq), and Cs₂CO₃ (437 mg, 1.34 mmol, 2.8eq). The vial was sealed and evacuated/backfilled with nitrogen (3×).Dioxane (2.5 mL) was added, and the reaction was flushed again withnitrogen (3×). Following the addition of pyrrolidine (96.1 μL, 1.15mmol, 2.4 eq), the reaction was stirred at 80° C. for 4 h. It was thencooled to room temperature, filtered through Celite with CH₂Cl₂, andconcentrated in vacuo. Purification by silica gel chromatography (0-40%EtOAc/toluene, linear gradient) afforded the N-acetyl leuco-dye (112 mg,64%) as an off-white solid. The intermediate leuco-dye (112 mg, 0.308mmol) was taken up in a mixture of CH₂Cl₂ (9 mL) and water (1 mL). DDQ(105 mg, 0.462 mmol, 1.5 eq) was added, and the reaction was stirred atroom temperature for 2 h. The crude reaction mixture was then depositedonto Celite and concentrated to dryness. Silica gel chromatography(0-20% MeOH/CH₂Cl₂, linear gradient, with constant 1% v/v AcOH additive;dry load with Celite) followed by reverse phase HPLC (10-50% MeCN/H₂O,linear gradient, with constant 0.1% v/v TFA additive) afforded 127 mg(95%) of the title compound as a dark blue solid. ¹H NMR (CD₃OD, 400MHz) δ 7.77 (d, J=9.5 Hz, 2H), 7.25 (dd, J=9.4, 2.5 Hz, 2H), 6.81 (d,J=2.5 Hz, 2H), 3.82-3.66 (m, 8H), 2.22-2.13 (m, 8H); ¹³C NMR (CD₃OD, 101MHz) δ 156.6 (C), 150.4 (C), 135.5 (C), 135.3 (CH), 119.4 (CH), 98.0(CH), 50.9 (bs, CH₂), 26.2 (bs, CH₂); Analytical HPLC: t_(R)=10.4min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positiveion mode; detection at 650 nm); HRMS (ESI) calcd for C₂₀H₂₂N₃O[M]⁺320.1757, found 320.1763.

Example 30. 3,7-Bis(pyrrolidin-1-yl-d₈)phenoxazin-5-ium trifluoroacetate

The title compound (88%, dark blue solid) was prepared from10-acetyl-10H-phenoxazine-3,7-diyl bis(trifluoromethanesulfonate) andpyrrolidine-2,2,3,3,4,4,5,5-d₈ according to the procedure described forExample 29. ¹H NMR (CD₃OD, 400 MHz) δ 7.77 (d, J=9.4 Hz, 2H), 7.25 (dd,J=9.5, 2.5 Hz, 2H), 6.80 (d, J=2.5 Hz, 2H); Analytical HPLC: t_(R)=10.3min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positiveion mode; detection at 650 nm); HRMS (ESI) calcd for C₂₀H₆D₁₆N₃O[M]⁺336.2762, found 336.2765.

Example 31.2-(3,6-Bis(azetidin-1-yl-d₆)xanthylium-9-yl)-4-carboxybenzoate

2-(3,6-Bis(azetidin-1-yl-d₆)xanthylium-9-yl)-4-(tert-butoxycarbonyl)benzoate(Example 13; 102 mg, 0.195 mmol) was taken up in CH₂Cl₂ (2.5 mL), andtrifluoroacetic acid (0.5 mL) was added. The reaction was stirred atroom temperature for 6 h. Toluene (3 mL) was added; the reaction mixturewas concentrated to dryness and then azeotroped with MeOH three times toprovide the title compound as a red-brown solid (109 mg, 96%, TFA salt).Analytical HPLC and NMR indicated that the material was >95% pure anddid not require further purification prior to amide coupling. ¹H NMR(CD₃OD, 400 MHz) δ 8.40 (d, J=8.1 Hz, 1H), 8.37 (dd, J=8.2, 1.5 Hz, 1H),7.95-7.93 (m, 1H), 7.06 (d, J=9.2 Hz, 2H), 6.60 (dd, J=9.2, 2.2 Hz, 2H),6.54 (d, J=2.2 Hz, 2H); Analytical HPLC: t_(R)=9.9 min, >99% purity (5μL injection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/vTFA additive; 20 min run; 1 mL/min flow; ESI; positive ion mode;detection at 550 nm); HRMS (ESI) calcd for C₂₇H₁₁D₁₂N₂O₅ [M+H]⁺467.2355,found 467.2354.

Example 32. 4-Carboxy-2-(3,6-di(pyrrolidin-1-yl)xanthylium-9-yl)benzoate

The title compound (99%, dark red-purple solid, TFA salt) was preparedfrom4-(tert-butoxycarbonyl)-2-(3,6-di(pyrrolidin-1-yl)xanthylium-9-yl)benzoate(Example 14) according to the procedure described for Example 32. ¹H NMR(CD₃OD, 400 MHz) δ 8.42 (d, J=8.2 Hz, 1H), 8.39 (dd, J=8.2, 1.5 Hz, 1H),7.99-7.96 (m, 1H), 7.11 (d, J=9.4 Hz, 2H), 6.92 (dd, J=9.4, 2.3 Hz, 2H),6.84 (d, J=2.3 Hz, 2H), 3.68-3.56 (m, 8H), 2.21-2.07 (m, 8H); ¹³C NMR(CD₃OD, 101 MHz) δ 167.7 (C), 167.4 (C), 160.0 (C), 158.9 (C), 156.2(C), 136.1 (C), 135.9 (C), 135.5 (C), 132.8 (CH), 132.31 (CH), 132.28(CH), 132.0 (CH), 116.4 (CH), 114.9 (C), 97.8 (CH), 50.0 (CH₂), 26.2(CH₂); Analytical HPLC: t_(R)=10.8 min, >99% purity (5 μL injection;10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive;20 min run; 1 mL/min flow; ESI; positive ion mode; detection at 550 nm);HRMS (ESI) calcd for C₂₉H₂₇N₂O₅ [M+H]⁺483.1914, found 483.1919.

Example 33.2-(3,6-Bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)-4-carboxybenzoate

The title compound (97%, dark red-purple solid, TFA salt) was preparedfrom2-(3,6-bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)-4-(tert-butoxycarbonyl)benzoate(Example 15) according to the procedure described for Example 32. ¹H NMR(CD₃OD, 400 MHz) δ 8.41 (d, J=8.2 Hz, 1H), 8.38 (dd, J=8.2, 1.5 Hz, 1H),7.99-7.96 (m, 1H), 7.11 (d, J=9.4 Hz, 2H), 6.91 (dd, J=9.3, 2.3 Hz, 2H),6.83 (d, J=2.3 Hz, 2H); Analytical HPLC: t_(R)=10.8 min, >99% purity (5μL injection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/vTFA additive; 20 min run; 1 mL/min flow; ESI; positive ion mode;detection at 550 nm); HRMS (ESI) calcd for C₂₉H₁₁D₁₆N₂O₅ [M+H]⁺499.2919,found 499.2922.

Example 34.2-(3,7-Bis(azetidin-1-yl-d₆)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-carboxybenzoate

The title compound (98%, green solid, TFA salt) was prepared from2-(3,7-bis(azetidin-1-yl-d₆)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(tert-butoxycarbonyl)benzoate(Example 16) according to the procedure described for Example 32. ¹H NMR(CD₃OD, 400 MHz) δ 8.29-8.25 (m, 2H), 7.80 (t, J=1.0 Hz, 1H), 6.90 (d,J=2.6 Hz, 2H), 6.87 (d, J=9.2 Hz, 2H), 6.33 (dd, J=9.2, 2.6 Hz, 2H),0.60 (s, 3H), 0.53 (s, 3H); Analytical HPLC: t_(R)=10.9 min, 97.8%purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 650 nm); HRMS (ESI) calcd for C₂₉H₁₇D₁₂N₂O₄Si[M+H]⁺509.2644, found 509.2648.

Example 35.4-Carboxy-2-(5,5-dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (99%, blue-green solid, TFA salt) was prepared from4-(tert-butoxycarbonyl)-2-(5,5-dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate(Example 17) according to the procedure described for Example 32. ¹H NMR(CD₃OD, 400 MHz) δ 8.34 (d, J=8.2 Hz, 1H), 8.29 (dd, J=8.2, 1.7 Hz, 1H),7.82 (d, J=1.6 Hz, 1H), 7.20 (d, J=2.7 Hz, 2H), 6.94 (d, J=9.4 Hz, 2H),6.61 (dd, J=9.5, 2.7 Hz, 2H), 3.73-3.58 (m, 8H), 2.16-2.05 (m, 8H), 0.64(s, 3H), 0.57 (s, 3H); ¹³C NMR (CD₃OD, 101 MHz) δ 170.3 (C), 168.0 (C),167.5 (C), 152.9 (C), 149.3 (C), 142.5 (C), 141.6 (CH), 136.1 (C), 135.2(C), 132.5 (CH), 132.4 (CH), 131.0 (CH), 129.1 (C), 122.6 (CH), 115.7(CH), 49.9 (CH₂), 26.1 (CH₂), −0.8 (CH₃), −1.8 (CH₃); Analytical HPLC:t_(R)=11.5 min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd forC₃₁H₃₃N₂O₄Si [M+H]⁺525.2204, found 525.2214.

Example 36.4-Carboxy-2-(5,5-dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (˜100%, blue-green solid, TFA salt) was prepared from4-(tert-butoxycarbonyl)-2-(5,5-dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate(Example 18) according to the procedure described for Example 32. ¹H NMR(CD₃OD, 400 MHz) δ 8.34 (d, J=8.1 Hz, 1H), 8.29 (dd, J=8.2, 1.6 Hz, 1H),7.83-7.81 (m, 1H), 7.19 (d, J=2.7 Hz, 2H), 6.94 (d, J=9.5 Hz, 2H), 6.60(dd, J=9.5, 2.7 Hz, 2H), 0.64 (s, 314), 0.57 (s, 3H); Analytical HPLC:t_(R)=11.5 min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd forC₃₁H₁₇D₁₆N₂O₄Si [M+H]⁺541.3208, found 541.3213.

Example 37.2-(3,6-Bis(azetidin-1-yl-d₆)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-carboxybenzoate

The title compound (˜100%, blue-green solid) was prepared from2-(3,6-bis(azetidin-1-yl-d₆)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-(tert-butoxycarbonyl)benzoate(Example 19) according to the procedure described for Example 32. ¹H NMR(CD₃OD, 400 MHz) δ 8.34 (d, J=8.2 Hz, 1H), 8.33-8.28 (m, 1H), 7.86-7.82(m, 1H), 6.93 (d, J=9.1 Hz, 2H), 6.82 (d, J=2.3 Hz, 2H), 6.38 (dd,J=9.1, 2.2 Hz, 2H), 1.82 (s, 3H), 1.70 (s, 3H); MS (ESI) calcd forC₃₀H₁₇D₁₂N₂O₄ [M+H]⁺493.3, found 493.2.

Example 38.4-Carboxy-2-(10,10-dimethyl-3,6-di(pyrrolidin-1-yl)anthracen-9-ylium-9(10H)-yl)benzoate

The title compound (˜100%, blue solid) was prepared from4-(tert-butoxycarbonyl)-2-(10,10-dimethyl-3,6-di(pyrrolidin-1-yl)anthracen-9-ylium-9(10H)-yl)benzoate(Example 20) according to the procedure described for Example 32. ¹H NMR(CD₃OD, 400 MHz) δ 8.38-8.28 (m, 2H), 7.92-7.83 (m, 1H), 7.10 (d, J=2.3Hz, 2H), 6.99 (d, J=9.3 Hz, 2H), 6.67 (dd, J=9.2, 2.3 Hz, 2H), 3.71-3.59(m, 8H), 2.17-2.07 (m, 8H), 1.88 (s, 3H), 1.76 (s, 3H); HRMS (ESI) calcdfor C₃H₃₃N₂O₄ [M+H]⁺509.2435, found 509.2435.

Example 39.4-Carboxy-2-(10,10-dimethyl-3,6-bis(pyrrolidin-1-yl-d₈)anthracen-9-ylium-9(10H)-yl)benzoate

The title compound (˜100%, blue solid) was prepared from4-(tert-butoxycarbonyl)-2-(10,10-dimethyl-3,6-bis(pyrrolidin-1-yl-d₈)anthracen-9-ylium-9(10H)-yl)benzoate(Example 21) according to the procedure described for Example 32. ¹H NMR(CD₃OD, 400 MHz) δ 8.36 (dd, J=8.2, 0.4 Hz, 1H), 8.33 (dd, J=8.2, 1.6Hz, 1H), 7.88-7.86 (m, 1H), 7.10 (d, J=2.4 Hz, 2H), 6.99 (d, J=9.3 Hz,2H), 6.67 (dd, J=9.3, 2.4 Hz, 2H), 1.88 (s, 3H), 1.76 (s, 3H); MS (ESI)calcd for C₃₂H₁₇D₁₆N₂O₄ [M+H]⁺525.3, found 525.3.

Example 40.2-(3,6-Bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyebenzoate

2-(3,6-Bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)-4-carboxybenzoate(Example 34; 25 mg, 40.8 μmol, TFA salt) was combined with DSC (25.1 mg,97.9 μmol, 2.4 eq) in DMF (1 mL). After adding Et₃N (34.1 μL, 0.245mmol, 6 eq) and DMAP (0.5 mg, 4.1 μmol, 0.1 eq), the reaction wasstirred at room temperature for 1 h. Purification of the crude reactionmixture by reverse phase HPLC (10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive) afforded 20.8 mg (72%, TFA salt) of thetitle compound as a dark red-purple solid. ¹H NMR (DMSO-d₆, 400 MHz) δ8.49 (dd, J=8.3, 1.8 Hz, 1H), 8.43 (d, J=8.2 Hz, 1H), 8.15 (d, J=1.8 Hz,1H), 7.11 (d, J=9.4 Hz, 2H), 6.91 (dd, J=9.3, 2.4 Hz, 2H), 6.83 (d,J=2.3 Hz, 2H), 2.91 (s, 4H); Analytical HPLC: t_(R)=11.4 min, >99%purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 550 nm); HRMS (ESI) calcd for C₃₃H₁₄D₁₆N₃O₇[M+H]⁺596.3083, found 596.3089.

Example 41.2-(5,5-Dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate

The title compound (87%, blue-green solid) was prepared from4-carboxy-2-(5,5-dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate(Example 36) according to the procedure described for Example 41. ¹H NMR(CDCl₃, 400 MHz) δ 8.27 (dd, J=8.0, 1.4 Hz, 1H), 8.07 (dd, J=8.0, 0.8Hz, 1H), 8.00 (dd, J=1.3, 0.8 Hz, 1H), 6.79 (d, J=2.7 Hz, 2H), 6.74 (d,J=8.8 Hz, 2H), 6.43 (dd, J=8.9, 2.7 Hz, 2H), 3.35-3.25 (m, 8H), 2.88 (s,4H), 2.04-1.94 (m, 8H), 0.64 (s, 3H), 0.58 (s, 3H); ¹³C NMR (CDCl₃, 101MHz) δ 169.6 (C), 168.9 (C), 161.2 (C), 155.8 (C), 147.0 (C), 136.9 (C),132.0 (C), 130.6 (CH), 130.1 (C), 129.7 (C), 128.3 (CH), 126.8 (CH),126.2 (CH), 116.1 (CH), 113.2 (CH), 92.9 (C), 47.6 (CH₂), 25.8 (CH₂),25.6 (CH₂), 0.4 (CH₃), −1.1 (CH₃); Analytical HPLC: t_(R)=12.2 min,98.6% purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positiveion mode; detection at 650 nm); HRMS (ESI) calcd for C₃₅H₃₆N₃O₆Si[M+H]⁺622.2368, found 622.2369.

Example 42.2-(5,5-Dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate

The title compound (92%, blue-green solid) was prepared from4-carboxy-2-(5,5-dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate(Example 37) according to the procedure described for Example 41. ¹H NMR(CDCl₃, 400 MHz) δ 8.27 (dd, J=8.0, 1.4 Hz, 1H), 8.07 (dd, J=8.0, 0.6Hz, 1H), 8.01 (dd, J=1.4, 0.8 Hz, 1H), 6.78 (d, J=2.8 Hz, 2H), 6.73 (d,J=8.8 Hz, 2H), 6.42 (dd, J=8.8, 2.8 Hz, 2H), 2.89 (s, 4H), 0.64 (s, 3H),0.58 (s, 3H); Analytical HPLC: t_(R)=12.1 min, 98.8% purity (5 μLinjection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 20 min run; 1 mL/min flow; ESI; positive ion mode; detectionat 650 nm); HRMS (ESI) calcd for C₃₅H₂₀D₁₆N₃O₆Si [M+H]⁺638.3372, found638.3380.

Example 43.2-(3,6-Bis(azetidin-1-yl-d₆)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate

2-(3,6-Bis(azetidin-1-yl-d₆)xanthylium-9-yl)-4-carboxybenzoate (Example32; 25 mg, 43.1 μmol) was combined with DSC (26.5 mg, 0.103 mmol, 2.4eq) in DMF (1 mL). After adding Et₃N (36.0 μL, 0.258 mmol, 6 eq) andDMAP (0.5 mg, 4.3 μmol, 0.1 eq), the reaction was stirred at roomtemperature for 30 min. A solution of2-(2-((6-chlorohexyl)oxy)ethoxy)-ethanamine (“HaloTag(02)amine,” 43.6mg, 0.129 mmol, 3 eq) in DMF (500 μL) was then added. The reaction wasstirred an additional 2 h at room temperature. Purification of the crudereaction mixture by reverse phase HPLC (20-60% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive) afforded 22 mg (66%, TFAsalt) of the title compound as a dark red solid. ¹H NMR (CD₃OD, 400 MHz)δ 8.78 (t, J=5.5 Hz, 1H), 8.39 (d, J=8.2 Hz, 1H), 8.20 (dd, J=8.2, 1.8Hz, 1H), 7.80 (d, J=1.7 Hz, 1H), 7.06 (d, J=9.2 Hz, 2H), 6.60 (dd,J=9.2, 2.2 Hz, 2H), 6.55 (d, J=2.2 Hz, 2H), 3.69-3.55 (m, 8H), 3.53 (t,J=6.6 Hz, 2H), 3.43 (t, J=6.5 Hz, 2H), 1.76-1.67 (m, 2H), 1.55-1.46 (m,2H), 1.45-1.28 (m, 4H); Analytical HPLC: t_(R)=12.3 min, >99% purity (5μL injection; 10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/vTFA additive; 20 min run; 1 mL/min flow; ESI; positive ion mode;detection at 550 nm); HRMS (ESI) calcd for C₃₇H₃₁D₁₂ClN₃O₆[M+H]⁺672.3588, found 672.3590.

Example 44.4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,6-di(pyrrolidin-1-yl)xanthylium-9-yl)benzoate

The title compound (58%, dark red-purple solid, TFA salt) was preparedfrom 4-carboxy-2-(3,6-di(pyrrolidin-1-yl)xanthylium-9-yl)benzoate(Example 33) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine according tothe procedure described for Example 44. ¹H NMR (CD₃OD, 400 MHz) δ 8.76(t, J=5.3 Hz, 1H), 8.40 (d, J=8.2 Hz, 1H), 8.21 (dd, J=8.3, 1.8 Hz, 1H),7.83 (d, J=1.7 Hz, 1H), 7.12 (d, J=9.3 Hz, 2H), 6.91 (dd, J=9.3, 2.3 Hz,2H), 6.84 (d, J=2.3 Hz, 2H), 3.71-3.54 (m, 16H), 3.51 (d, J=6.6 Hz, 2H),3.43 (t, J=6.5 Hz, 2H), 2.20-2.05 (m, 8H), 1.76-1.66 (m, 2H), 1.54-1.45(m, 2H), 1.45-1.26 (m, 4H); Analytical HPLC: t_(R)=13.2 min, 98.4%purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positive ionmode; detection at 550 nm); HRMS (ESI) calcd for C₃₉H₄₇ClN₃O₆[M+H]⁺688.3148, found 688.3156.

Example 45.2-(3,6-Bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)-4-((2-(2-((6-chlorohexypoxy)ethoxy)ethyl)carbamoyl)benzoate

The title compound (63%, dark red-purple solid, TFA salt) was preparedfrom 2(3,6-bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)-4-carboxybenzoate(Example 34) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine according tothe procedure described for Example 44. ¹H NMR (CD₃OD, 400 MHz) δ 8.76(t, J=5.2 Hz, 1H), 8.40 (d, J=8.2 Hz, 1H), 8.21 (dd, J=8.3, 1.8 Hz, 1H),7.83 (d, J=1.7 Hz, 1H), 7.12 (d, J=9.3 Hz, 2H), 6.91 (dd, J=9.3, 2.3 Hz,2H), 6.84 (d, J=2.3 Hz, 2H), 3.70-3.54 (m, 8H), 3.52 (t, J=6.6 Hz, 2H),3.43 (t, J=6.5 Hz, 2H), 1.76-1.66 (m, 2H), 1.55-1.45 (m, 2H), 1.45-1.27(m, 4H); Analytical HPLC: t_(R)=13.1 min, 98.4% purity (5 μL injection;10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive;20 min run; 1 mL/min flow; ESI; positive ion mode; detection at 550 nm);HRMS (ESI) calcd for C₃₉H₃₁D₁₆ClN₃O₆ [M+H]⁺704.4152, found 704.4155.

Example 46.2-(3,7-Bis(azetidin-1-yl-d₆)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate

The title compound (79%, pale blue-green solid) was prepared from2-(3,7-bis(azetidin-1-yl-d₆)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-carboxybenzoate(Example 35) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine according tothe procedure described for Example 44. ¹H NMR (CDCl₃, 400 MHz) δ 7.98(d, J=7.9 Hz, 1H), 7.90 (dd, J=8.0, 1.4 Hz, 1H), 7.70-7.66 (m, 1H), 6.76(s, 1H), 6.75 (d, J=8.7 Hz, 2H), 6.66 (d, J=2.7 Hz, 2H), 6.26 (dd,J=8.6, 2.7 Hz, 2H), 3.67-3.59 (m, 6H), 3.56-3.53 (m, 2H), 3.50 (t, J=6.6Hz, 2H), 3.39 (t, J=6.7 Hz, 2H), 1.76-1.69 (m, 2H), 1.54-1.48 (m, 2H),1.44-1.26 (m, 4H), 0.63 (s, 3H), 0.57 (s, 3H); Analytical HPLC:t_(R)=13.2 min, 98.7% purity (5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd forC₃₉H₃₇D₁₂ClN₃O₅Si [M+H]⁺714.3878, found 714.3885.

Example 47.4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(5,5-dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

2-(5,5-Dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate(Example 42; 75 mg, 0.121 mmol) and2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine (“HaloTag(O2)amine,” 61.1 mg,0.181 mmol, 1.5 eq) were combined in DMF (3 mL), and DIEA (63.0 μL,0.362 mmol, 3 eq) was added. After stirring the reaction at roomtemperature for 1 h, it was diluted with saturated NaHCO₃ and extractedwith EtOAc (2×). The combined organic extracts were washed with waterand brine, dried over anhydrous MgSO₄, filtered, and evaporated.Purification of the crude product by silica gel chromatography (10-100%EtOAc/toluene, linear gradient) provided the title compound as a paleblue-green solid (64 mg, 73%). ¹H NMR (CDCl₃, 400 MHz) δ 7.99 (d, J=7.9Hz, 1H), 7.91 (dd, J=7.9, 1.4 Hz, 1H), 7.67-7.64 (m, 1H), 6.79 (d, J=2.7Hz, 2H), 6.75 (s, 1H), 6.75 (d, J=8.8 Hz, 2H), 6.39 (dd, J=8.9, 2.7 Hz,2H), 3.67-3.58 (m, 6H), 3.57-3.52 (m, 2H), 3.50 (t, J=6.7 Hz, 2H), 3.39(t, J=6.7 Hz, 2H), 3.35-3.24 (m, 8H), 2.05-1.93 (m, 8H), 1.77-1.68 (m,2H), 1.55-1.47 (m, 2H), 1.44-1.26 (m, 4H), 0.65 (s, 3H), 0.59 (s, 3H);Analytical HPLC: t_(R)=13.5 min, >99% purity (5 μL injection; 10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 650 nm); HRMS(ESI) calcd for C₄₁H₅₃ClN₃O₅Si [M+H]⁺730.3438, found 730.3442.

Example 48.4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(5,5-dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (77%, pale blue-green solid) was prepared from2-(5,5-dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate(Example 43) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine according tothe procedure described for Example 48. ¹H NMR (CDCl₃, 400 MHz) δ 7.99(dd, J=8.0, 0.5 Hz, 1H), 7.91 (dd, J=8.0, 1.4 Hz, 1H), 7.67-7.63 (m,1H), 6.78 (d, J=2.8 Hz, 2H), 6.747 (d, J=8.8 Hz, 2H), 6.744 (s, 1H),6.39 (dd, J=8.8, 2.8 Hz, 2H), 3.66-3.58 (m, 6H), 3.57-3.52 (m, 2H), 3.50(t, J=6.7 Hz, 2H), 3.39 (t, J=6.7 Hz, 2H), 1.77-1.68 (m, 2H), 1.55-1.47(m, 2H), 1.44-1.35 (m, 2H), 1.34-1.25 (m, 2H), 0.65 (s, 3H), 0.58 (s,3H); Analytical HPLC: t_(R)=13.4 min, >99% purity (5 μL injection;10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive;20 min run; 1 mL/min flow; ESI; positive ion mode; detection at 650 nm);HRMS (ESI) calcd for C₄₁H₃₇D₁₆ClN₃O₅Si [M+H]⁺746.4442, found 746.4449.

Example 49.2-(3,6-Bis(azetidin-1-yl-d₆)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate

Step 1: The procedure described for Example 41 was used to prepare2-(3,6-bis(azetidin-1-yl-d₆)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoatefrom2-(3,6-bis(azetidin-1-yl-d₆)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-carboxybenzoate(Example 38). MS (ESI) calcd for C₃₄H₂₀D₁₂N₃O₆ [M+H]⁺590.3, found 590.3.

Step 2: The title compound (25%, blue solid) was prepared from2-(3,6-bis(azetidin-1-yl-d₆)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate(Step 1) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine according to theprocedure described for Example 48. ¹H NMR (CDCl₃, 400 MHz) δ 8.02 (d,J=7.9 Hz, 1H), 7.97-7.91 (m, 1H), 7.44-7.39 (m, 1H), 6.70 (s, 1H), 6.57(d, J=2.4 Hz, 2H), 6.52 (d, J=8.6 Hz, 2H), 6.20 (dd, J=8.6, 2.4 Hz, 2H),3.63-3.48 (m, 10H), 3.38 (t, J=6.6 Hz, 2H), 1.83 (s, 3H), 1.78-1.69 (m,2H), 1.72 (s, 3H), 1.52-1.26 (m, 6H); Analytical HPLC: t_(R)=12.6min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positiveion mode; detection at 600 nm); MS (ESI) calcd for C₄₀H₃₇D₁₂ClN₃O₅[M+H]⁺698.4, found 698.3.

Example 50.4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(10,10-dimethyl-3,6-di(pyrrolidin-1-yl)anthracen-9-ylium-9(10H)-yl)benzoate

Step 1: The procedure described for Example 41 was used to prepare2-(10,10-dimethyl-3,6-di(pyrrolidin-1-yl)anthracen-9-ylium-9(10H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoatefrom4-carboxy-2-(10,10-dimethyl-3,6-di(pyrrolidin-1-yl)anthracen-9-ylium-9(10H)-yl)benzoate(Example 39). MS (ESI) calcd for C₃₆H₃₆N₃O₆ [M+H]⁺606.3, found 606.2.

Step 2: The title compound (11%, blue solid) was prepared from2-(10,10-dimethyl-3,6-di(pyrrolidin-1-yl)anthracen-9-ylium-9(10H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate(Step 1) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine according to theprocedure described for Example 48. ¹H NMR (CD₃OD, 400 MHz) δ 8.77-8.72(m, 1H), 8.35 (d, J=8.2 Hz, 1H), 8.16 (dd, J=8.3, 1.5 Hz, 1H), 7.77 (d,J=1.8 Hz, 1H), 7.10 (d, J=2.4 Hz, 2H), 7.00 (d, J=9.3 Hz, 2H), 6.67 (dd,J=9.2, 2.3 Hz, 2H), 3.72-3.54 (m, 16H), 3.51 (t, J=6.6 Hz, 2H), 3.43 (t,J=6.5 Hz, 2H), 2.20-2.05 (m, 8H), 1.87 (s, 3H), 1.77 (s, 3H), 1.74-1.66(m, 2H), 1.54-1.47 (m, 2H), 1.42-1.31 (m, 4H); Analytical HPLC:t_(R)=13.3 min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; detection at 600 nm); MS (ESI) calcd forC₄₂H₅₃ClN₃O₅ [M+H]⁺714.4, found 714.3.

Example 51.4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(10,10-dimethyl-3,6-bis(pyrrolidin-1-yl-d8)anthracen-9-ylium-9(10H)-yl)benzoate

Step 1: The procedure described for Example 41 was used to prepare2-(10,10-dimethyl-3,6-bis(pyrrolidin-1-yl-d₈)anthracen-9-ylium-9(10H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoatefrom4-carboxy-2-(10,10-dimethyl-3,6-bis(pyrrolidin-1-yl-d₈)anthracen-9-ylium-9(10H)-yl)benzoate(Example 40). MS (ESI) calcd for C₃₆H₂₀D₁₆N₃O₆ [M+H]⁺622.4, found 622.3.

Step 2: The title compound (31%, blue solid) was prepared from2-(10,10-dimethyl-3,6-bis(pyrrolidin-1-yl-d₈)anthracen-9-ylium-9(10H)-yl)-4-((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate(Step 1) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine according to theprocedure described for Example 48. ¹H NMR (CD₃OD, 400 MHz) δ 8.75 (t,J=5.1 Hz, 1H), 8.35 (d, J=8.2 Hz, 1H), 8.16 (dd, J=8.2, 1.9 Hz, 1H),7.77 (d, J=1.7 Hz, 1H), 7.10 (d, J=2.4 Hz, 2H), 7.00 (d, J=9.2 Hz, 2H),6.66 (dd, J=9.3, 2.4 Hz, 2H), 3.70-3.54 (m, 8H), 3.51 (t, J=6.6 Hz, 2H),3.43 (t, J=6.5 Hz, 2H), 1.86 (s, 3H), 1.76 (s, 3H), 1.75-1.67 (m, 2H),1.50 (p, J=6.8 Hz, 2H), 1.43-1.31 (m, 4H); Analytical HPLC: t_(R)=12.9min, 98.4% purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient,with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI;positive ion mode; detection at 600 nm); MS (ESI) calcd forC₄₂H₃₇D₁₆ClN₃O₅ [M+H]⁺730.5, found 730.4.

Example 52.4-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-di(pyrrolidin-1-yl)xanthylium-9-yl)benzoate

The title compound (66%, dark red-purple solid) was prepared from4-carboxy-2-(3,6-di(pyrrolidin-1-yl)xanthylium-9-yl)benzoate (Example33) and 6-((4-(aminomethyl)benzyl)oxy)-9H-purin-2-amine (“BG-NH₂”)according to the procedure described for Example 44. ¹H NMR (CD₃OD, 400MHz) δ 9.30 (t, J=6.0 Hz, 1H), 8.40 (d, J=8.2 Hz, 1H), 8.26 (s, 1H),8.22 (dd, J=8.3, 1.8 Hz, 1H), 7.84 (d, J=1.7 Hz, 1H), 7.51 (d, J=8.1 Hz,2H), 7.41 (d, J=8.1 Hz, 2H), 7.10 (d, J=9.4 Hz, 2H), 6.89 (dd, J=9.3,2.3 Hz, 2H), 6.83 (d, J=2.2 Hz, 2H), 5.62 (s, 2H), 4.60 (s, 2H),3.66-3.57 (m, 8H), 2.20-2.08 (m, 8H); Analytical HPLC: t_(R)=10.0min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positiveion mode; detection at 550 nm); HRMS (ESI) calcd for C₄₂H₃₉N₈O₅[M+H]⁺735.3038, found 735.3046.

Example 53.4-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-bis(pyrrolidin-1-yl-d₈)-9H-xanthen-9-ylium-9-yl)benzoate

The title compound (68%, dark red-purple solid) was prepared from2-(3,6-bis(pyrrolidin-1-yl-d₈)xanthylium-9-yl)-4-carboxybenzoate(Example 34) and 6-((4-(aminomethyl)benzyl)oxy)-9H-purin-2-amineaccording to the procedure described for Example 44. ¹H NMR (CD₃OD, 400MHz) δ 9.30 (t, J=5.9 Hz, 1H), 8.40 (d, J=8.2 Hz, 1H), 8.216 (s, 1H),8.215 (dd, J=8.2, 1.8 Hz, 1H), 7.84 (d, J=1.7 Hz, 1H), 7.50 (d, J=8.0Hz, 2H), 7.40 (d, J=7.9 Hz, 2H), 7.09 (d, J=9.3 Hz, 2H), 6.88 (dd,J=9.3, 2.3 Hz, 2H), 6.82 (d, J=2.3 Hz, 2H), 5.60 (s, 2H), 4.60 (s, 2H);Analytical HPLC: t_(R)=10.0 min, >99% purity (5 μL injection; 10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 550 nm); HRMS(ESI) calcd for C₄₂H₂₃D₁₆N₈O₅ [M+H]⁺751.4042, found 751.4054.

Example 54.4-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(5,5-dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate

The title compound (76%, blue solid) was prepared from2-(5,5-dimethyl-3,7-di(pyrrolidin-1-yl)dibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate(Example 42) and 6-((4-(aminomethyl)benzyl)oxy)-9H-purin-2-amineaccording to the procedure described for Example 48. ¹H NMR (CD₃OD, 400MHz) δ 8.04 (dd, J=8.1, 1.4 Hz, 1H), 8.00 (dd, J=8.0, 0.7 Hz, 1H), 7.83(s, 1H), 7.71-7.69 (m, 1H), 7.44 (d, J=8.2 Hz, 2H), 7.31 (d, J=8.2 Hz,2H), 6.86 (d, J=2.8 Hz, 2H), 6.69 (d, J=8.9 Hz, 2H), 6.42 (dd, J=8.9,2.8 Hz, 2H), 5.49 (s, 2H), 4.52 (s, 2H), 3.32-3.24 (m, 8H), 2.07-1.95(m, 8H), 0.62 (s, 3H), 0.55 (s, 3H); Analytical HPLC: t_(R)=10.5min, >99% purity (5 μL injection; 10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow; ESI; positiveion mode; detection at 650 nm); HRMS (ESI) calcd for C₄₄H₄₅N₈O₄Si[M+H]⁺777.3328, found 777.3339.

Example 55.4-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(5,5-dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(51H)-yl)benzoate

The title compound (78%, blue solid) was prepared from2-(5,5-dimethyl-3,7-bis(pyrrolidin-1-yl-d₈)dibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate(Example 43) and 6-((4-(aminomethyl)benzyl)oxy)-9H-purin-2-amineaccording to the procedure described for Example 48. ¹H NMR (CD₃OD, 400MHz) δ 8.02 (dd, J=8.0, 1.4 Hz, 1H), 7.98 (dd, J=8.0, 0.5 Hz, 1H), 7.81(s, 1H), 7.69-7.67 (m, 1H), 7.42 (d, J=8.2 Hz, 2H), 7.29 (d, J=8.1 Hz,2H), 6.84 (d, J=2.8 Hz, 2H), 6.67 (d, J=8.9 Hz, 2H), 6.40 (dd, J=8.9,2.8 Hz, 2H), 5.47 (s, 2H), 4.50 (s, 2H), 0.60 (s, 3H), 0.53 (s, 3H);Analytical HPLC: t_(R)=10.4 min, >99% purity (5 μL injection; 10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 minrun; 1 mL/min flow; ESI; positive ion mode; detection at 650 nm); HRMS(ESI) calcd for C₄₄H₂₉D₁₆N₈O₄Si [M+H]⁺793.4332, found 793.4341.

The invention claimed is:
 1. A compound selected from the followinggroup of compounds:


2. A compound selected from the following group of compounds:


3. A compound selected from the following group of compounds: